Methods and systems for detecting biological components

ABSTRACT

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

CROSS-REFERENCE

This application is a National Stage of International Application No.PCT/US2013/054517, filed Aug. 12, 2013, which application claimspriority to U.S. Provisional Application No. 61/682,707, filed Aug. 13,2012; and to U.S. Provisional Application No. 61/784,754, filed Mar. 14,2013; which applications are incorporated by reference herein in theirentireties and for all purposes.

GOVERNMENT RIGHTS

This invention was made with government support under grant nos.HG007233 and AR068129, awarded by the National Institutes of Health, andgrant no. DBI1253293, awarded by the National Science Foundation. Thegovernment has certain rights in the invention.

INTRODUCTION

Biological samples from a subject often contain a large number ofdifferent components. For example, a sample of a subject's blood maycontain free floating DNA and RNA, circulating cells, and many othercomponents. The number and diversity of such components in a biologicalsample often complicates or prevents the accurate identification and/orquantification of specific components of interest within the sample,which would enable the diagnosis or monitoring of a condition in thesubject, such as cancer.

For instance, circulating tumor cells (CTCs) are cells shed from tumorsthat enter into a subject's blood stream. Once in the blood, these cellscan circulate through the subject's body, where they can invade othertissues and grow new tumors. CTCs are thus implicated in metastasis,which is the primary cause of death in subjects with cancer. Efforts tocount CTCs have been hampered by the fact that CTCs are extremelydifficult to detect: they are exceptionally rare, and may be difficultto distinguish from healthy cells. Current approaches for detecting CTCsrely on immunoassays, in which antibodies are used to target specificbiomarkers on the surfaces of the CTCs. However, such approaches havelimitations in sensitivity and/or specificity, leading to many healthycells being mischaracterized as cancerous, and many cancer cells beingmissed in the analysis.

SUMMARY

Methods for the detection of components from biological samples areprovided. In certain aspects, the methods may be used to detect and/orquantify specific components in a biological sample, such as tumor cells(e.g., circulating tumor cells, or CTCs). Systems and devices for use inpracticing methods of the invention are also provided.

Methods of the present disclosure include methods for the detection ofcells in a biological sample, such as tumor cells. Using microfluidics,components of the biological sample may be encapsulated intomicrodroplets, which are tiny spheres of solution generally ranging from0.1 to 1000 μm in diameter, which may be used to encapsulate cells,polynucleotides, polypeptides, and other components. The componentsencapsulated in each microdroplet may be assayed, as described morefully herein.

Aspects of the methods may include encapsulating in a microdroplet acell obtained from a subject's blood sample, wherein at least one cellis present in the microdroplet; lysing the cell; introducing polymerasechain reaction (PCR) reagents, a detection component, and a plurality ofPCR primers into the microdroplet and incubating the microdroplet underconditions allowing for PCR amplification to produce PCR amplificationproducts, wherein the plurality of PCR primers include one or moreprimers that hybridizes to one or more oligonucleotides (e.g.,oncogenes); and detecting the presence or absence of the PCRamplification products by detection of the detection component, whereindetection of the detection component indicates the presence of PCRamplification products. In certain aspects, the step of lysing the cellinvolves introducing a lysing agent into the microdroplet and incubatingthe microdroplet under conditions effective for cell lysis. The methodsmay include determining the number of circulating tumor cells (CTCs)present in a sample of the subject's blood, based at least in part onthe number of microdroplets in which PCR products were detected. Inother aspects, the methods may include determining the number of tumorcells present in a solid tissue sample from the subject, based at leastin part on the number of microdroplets in which PCR products weredetected.

In other aspects, the methods for the detection of cells includeencapsulating a plurality of cells in a plurality of microdroplets underconditions in which a majority of microdroplets contain zero or onecell, wherein the plurality of cells are obtained from a subject's bloodsample; enriching the plurality of microdroplets for microdropletscontaining one cell; lysing the cell; introducing polymerase chainreaction (PCR) reagents, a detection component, and a plurality of PCRprimers into the plurality of microdroplets and incubating the pluralityof microdroplets under conditions allowing for PCR amplification toproduce PCR amplification products, wherein the plurality of PCR primersinclude one or more primers that each hybridize to one or moreoligonucleotides (e.g., oncogenes); detecting the presence or absence ofthe PCR amplification products by detection of the detection component,wherein detection of the detection component indicates the presence ofthe PCR amplification products; and determining the number of cellspresent in the sample of the subject's blood based at least in part onthe number of microdroplets in which the PCR amplification products weredetected; wherein one or more steps are performed under microfluidiccontrol. In certain aspects, the cells are tumor cells, and theplurality of PCR primers include one or more primers that each hybridizeto one or more oncogenes. The step of lysing the cell may involveintroducing a lysing agent into the microdroplet and incubating themicrodroplet under conditions effective for cell lysis.

Methods of the present disclosure also include methods for genotypingcells, including tumor cells. In certain aspects, the methods forgenotyping cells include encapsulating in a microdroplet a cell obtainedfrom a biological sample from the subject, wherein one cell is presentin the microdroplet; introducing a lysing agent into the microdropletand incubating the microdroplet under conditions effective for celllysis; introducing polymerase chain reaction (PCR) reagents and aplurality PCR primers into the microdroplet, and incubating themicrodroplet under conditions allowing for PCR amplification to producePCR amplification products; introducing a plurality of probes into themicrodroplet, wherein the probes hybridize to one or more mutations ofinterest and fluoresce at different wavelengths; and detecting thepresence or absence of specific PCR amplification products by detectionof fluorescence of a probe, wherein detection of fluorescence indicatesthe presence of the PCR amplification products; wherein one or more ofsteps are performed under microfluidic control. The plurality of probesmay include one or more TaqMan® probes.

Methods of the present disclosure also include methods for the detectionof cancer, the methods including encapsulating in a microdropletoligonucleotides obtained from a biological sample from the subject,wherein at least one oligonucleotide is present in the microdroplet;introducing polymerase chain reaction (PCR) reagents, a detectioncomponent, and a plurality of PCR primers into the microdroplet andincubating the microdroplet under conditions allowing for PCRamplification to produce PCR amplification products, wherein theplurality of PCR primers include one or more primers that each hybridizeto one or more oncogenes; and detecting the presence or absence of thePCR amplification products by detection of the detection component,wherein detection of the detection component indicates the presence ofthe PCR amplification products. The detection of cancer in the subjectmay be based upon the presence of PCR amplification products for one ormore oncogenes.

In other aspects, the methods of the present disclosure includeencapsulating in a microdroplet an oligonucleotide obtained from abiological sample obtained from a subject, wherein at least oneoligonucleotide is present in the microdroplet; introducing polymerasechain reaction (PCR) reagents, a detection component, and a plurality ofPCR primers into the microdroplet and incubating the microdroplet underconditions allowing for PCR amplification to produce PCR amplificationproducts; and detecting the presence or absence of the PCR amplificationproducts by detection of the detection component, wherein detection ofthe detection component indicates the presence of PCR amplificationproducts; wherein one or more steps are performed under microfluidiccontrol.

In practicing the subject methods, several variations may be employed.For example, a wide range of different PCR-based assays may be employed,such as quantitative PCR (qPCR). The number and nature of primers usedin such assays may vary, based at least in part on the type of assaybeing performed, the nature of the biological sample, and/or otherfactors. In certain aspects, the number of primers that may be added toa microdroplet may be 1 to 100 or more, and/or may include primers todetect from about 1 to 100 or more different genes (e.g., oncogenes). Inaddition to, or instead of, such primers, one or more probes (e.g.,TaqMan® probes) may be employed in practicing the subject methods.

The microdroplets themselves may vary, including in size, composition,contents, and the like. Microdroplets may generally have an internalvolume of about 0.001 to 1000 picoliters or more. Further, microdropletsmay or may not be stabilized by surfactants and/or particles.

The means by which reagents are added to a microdroplet may varygreatly. Reagents may be added in one step or in multiple steps, such as2 or more steps, 4 or more steps, or 10 or more steps. In certainaspects, reagents may be added using techniques including dropletcoalescence, picoinjection, multiple droplet coalescence, and the like,as shall be described more fully herein. In certain embodiments,reagents are added by a method in which the injection fluid itself actsas an electrode. The injection fluid may contain one or more types ofdissolved electrolytes that permit it to be used as such. Where theinjection fluid itself acts as the electrode, the need for metalelectrodes in the microfluidic chip for the purpose of adding reagentsto a droplet may be obviated. In certain embodiments, the injectionfluid does not act as an electrode, but one or more liquid electrodesare utilized in place of metal electrodes.

Various ways of detecting the absence or presence of PCR products may beemployed, using a variety of different detection components. Detectioncomponents of interest include, but are not limited to, fluorescein andits derivatives; rhodamine and its derivatives; cyanine and itsderivatives; coumarin and its derivatives; Cascade Blue and itsderivatives; Lucifer Yellow and its derivatives; BODIPY and itsderivatives; and the like. Exemplary fluorophores includeindocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5,Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, AlexaFluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluoresceinisothiocyanate (FITC), carboxy-fluorescein (FAM), phycoerythrin,rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine(TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen,RiboGreen, and the like. Detection components may include beads (e.g.,magnetic or fluorescent beads, such as Luminex beads) and the like. Incertain aspects, detection may involve holding a microdroplet at a fixedposition during thermal cycling so it can be repeatedly imaged. Suchrepeated imaging may involve the use of a Megadroplet Array, as shall bedescribed more fully herein. In certain aspects, detection may involvefixing and/or permeabilizing one or more cells in one or moremicrodroplets.

Suitable subjects for the methods disclosed herein include mammals,e.g., humans. The subject may be one that exhibits clinicalpresentations of a disease condition, or has been diagnosed with adisease. In certain aspects, the subject may be one that has beendiagnosed with cancer, exhibits clinical presentations of cancer, or isdetermined to be at risk of developing cancer due to one or more factorssuch as family history, environmental exposure, genetic mutation(s),lifestyle (e.g., diet and/or smoking), the presence of one or more otherdisease conditions, and the like.

Microfluidic systems and devices are also provided by the presentdisclosure. In certain aspects, the microfluidic devices include a cellloading region to encapsulate a cell to be analyzed in a microdroplet; afirst chamber in fluidic communication with the cell loading region, thefirst chamber having a means for adding a first reagent to themicrodroplet, and a heating element; a second chamber in fluidiccommunication with the first chamber, the second chamber having a meansfor adding a second reagent to the microdroplet, and a heating element,wherein the heating element may heat the microdroplet at one or moretemperatures; and a detection region, in fluidic communication with thesecond chamber, which detects the presence or absence of reactionproducts from the first or second chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 is a simplified depiction of a microfluidic system of the instantdisclosure. In the depicted system, the microfluidic system may be usedfor detecting and/or genotyping a component of a biological sample. Asapplied to the detection of tumor cells in this particular system,nucleated blood cells are encapsulated into individual droplets using anencapsulation device (left). The droplets are injected with a lysisbuffer and incubated at 37° C. to accelerate cell lysis. They areinjected with PCR mix containing primers targeting characteristiconcogenic mutations (center). The droplets are flowed through a channelsnaking over zones maintained at 65° C. and 95° C. As the droplets movethrough the zones, their temperature cycles, as needed for PCR. Duringthis PCR reaction, if a droplet contains a genome of a tumor cell with amutation for which the primers are designed to detect, amplificationwill be initiated, producing a fluorescent output that turns the dropletfluorescent. The droplets are then optically scanned using flowcytometry and sorted using droplet sorting to recover them (right). Thedroplets may be stored or used for further analysis, such as beingsubjected to sequencing (e.g., used as input for a next-gen sequencer,or provided to a sequencing facility).

FIG. 2, Panels A-E depict single cells enclosed in microdroplets, usinga fluorescence assay. Yeast cells (black specks) enter from the far leftand are encapsulated into drops, shown at low (4× objective; Panel A)and high magnification (10× objective; Panel B). The drops are incubatedto allowing the yeast to secrete their product (Panel C); this producesa fluorescent compound in the drops, so that drops containing efficientproducers quickly become fluorescent (Panel D). The drops are thensorted to extract the most efficient yeast using a microfluidic sorter(Panel E). The scale bars denote 80 mm.

FIG. 3 depicts digital detection of BRAF using a TaqMan® PCR probelabeled with the fluorophore FAM that is complementary to an ampliconfrom a portion of the human BRAF gene. Fluorescent drops indicateamplification of the BRAF gene from purified human genomic DNA, whilenon-fluorescent drops were devoid of the gene.

FIG. 4, Panels A-B depict a binary PCR reaction to detect CTCs. Panel A:Forward and reverse primers are encapsulated in the drops that target anoncogenic sequence. If the oncogenic sequence is present, the PCRreaction produces double-stranded PCR products (Panel A, upper),whereas, if it is not, no products are produced (Panel A, lower). Anintercalating stain (e.g., SybrGreen) may also be present in the drop.Panel B: If double-stranded products are produced, the dye intercalatesinto them, becoming fluorescent, and turning the drop fluorescent (PanelB, upper); by contrast, if no double-stranded products are produced, thedye remains non-fluorescent, producing a dim drop (Panel B, lower).

FIG. 5 is an optical microscopy image of massively parallel dropformation in a serial bisection device. DI water that does not containcells is injected from the left. The solution flowing in along the topand bottom arrows is HFE-7500 fluorocarbon oil with a fluorocarbonsurfactant at 2% by weight. After serial bisection, the resulting dropsshown to the far right are 25 μm in diameter.

FIG. 6 is a schematic microfluidic device and data showing procedure fordroplet-based detection of CTCs. Blood cells and rare CTCs areencapsulated in microdrops with lysis buffer containing Proteinase K.The drops are incubated at 55° C. to lyse cells and digest cellularproteins. Drops are then split to a size optimal for imaging, and theProteinase K is heat-inactivated. The drops are then picoinjected withPCR reagents and TaqMan® probes, followed by thermocycling and imagingon a Megadroplet Array. CTCs are identified based on the presence ofCTC-specific transcripts, detected by multiplexed TaqMan® probefluorescence.

FIG. 7 shows relief of cell lysate-mediated inhibition of RT-PCR byproteinase K treatment. Increasing concentrations of cells were eithertreated with proteinase K and lysis buffer or lysis buffer only. Cellswere then incubated at 55° C. followed by 95° C. Whole cell lysates wereadded directly to RT-PCR reactions at several drop relevantconcentrations. Strong relief of lysate inhibition on PCR was seen atfinal cell concentrations of 1 cell per 200 pL in Proteinase K treatedlysates but not in lysis buffer only lysates. PCR products arevisualized on an ethidium bromide stained agarose gel.

FIG. 8, Panels 1-3 show an integrated microfluidic system for cellencapsulation/dilution, lysis and drop splitting (center image). Panel1: Co-flow module relies on laminar flow of Proteinase K containinglysis buffer and cell suspension solutions to encapsulate cells in dropswithout premature lysis or mixing of cells prior to drop formation; alaminar flow boundary is just visible between the cell and lysis bufferstreams. Panel 2: Drops containing cells flow through a 55° C.incubation channel for 20 minutes to lyse cells and digest inhibitoryproteins. Panel 3: Drops are split to allow for efficient picoinjectionof 2× RT-PCR reagents and imaging on the droplet array.

FIG. 9, Panels A-C show TaqMan® RT-PCR in drops following picoinjection.Drops containing a limiting dilution of total RNA from the prostatecancer cell line PC3 were injected with an equal volume of 2× RT-PCRreagents and a TaqMan® probe targeting EpCAM, (Panel A). Followingpicoinjection, drops were thermocycled and imaged for fluorescence,(Panel B). The number of fluorescent drops was found to be in agreementwith the prediction of a Poisson distribution, demonstrating adequatesensitivity to detect single transcript molecules in drops. Panel C: Tofurther confirm the results, the drops from Panel B were chemicallyruptured and their contents run on an agarose gel to observe thepresence of PCR products in negative control drops that were injectedwithout RT-PCR enzymes (−) and experimental drops that received both RTand Taq (+). Both control reactions performed in a tube with nopicoinjection and picoinjected reactions produced bands of similarintensity, demonstrating that the reaction efficiency was comparable.White stars mark picoinjected drops.

FIG. 10 shows detection of EpCAM transcripts from droplet encapsulatedMCF7 breast cancer cells. Using the device depicted in FIG. 8, Panels1-3, MCF7 cells were encapsulated in drops, lysed and drops were split.Lysate containing drops were then picoinjected with RT-PCR reagents andTaqMan® probes. Drops were then thermocycled and imaged forfluorescence. Brightfield and fluorescent channels are shown merged.

FIG. 11 depicts digital droplet RT-PCR multiplexing with TaqMan® probes.Limiting dilutions of total RNA from both Raji cells (B-lymphocytes) andPC3 prostate cancer cells were encapsulated in drops together withRT-PCR reagents and TaqMan® probes specific to CD45 (blue), CD44 (red)and EpCAM (green). Orange drops indicate the presence of both CD44 andEpCAM transcripts detected by a multiplex reaction. Other probemultiplexing combinations have also been seen (data not shown).Fluorescent channels are shown individually as a magnified inset for thedashed box region.

FIG. 12, Panels A-C show a schematic illustration of a device forperforming multiplexed qPCR analysis on cells individually. The deviceconsists of an array of about 10 million traps indented into a PDMSchannel that sits above a thermal system (Panel A). The height of themicrofluidic channel is smaller than the diameter of the drops, causingdrops to adopt a flattened pancake shape. When a drop flows over anunoccupied indentation, it adopts a lower, more energetically favorable,radius of curvature, leading to a force that pulls the drop entirelyinto the trap (Panel B). By flowing drops as a close pack, it is ensuredthat all traps on the array are occupied, as illustrated in Panel C. Theentire device is thermal cycled and imaged between cycles using amicroarray scanner.

FIG. 13 depicts a Megadroplet Array for multiplexed qPCR analysis, ofthe type depicted in FIG. 12, Panels A-C. Drops are pipetted and sealedin a clear glass/epoxy chamber and fixed in place using amicrofabricated well array (top). The entire chip is clamped to a metalblock and thermocycled using Peltier heaters under the copper blocks.Thermometers, a heat sink, a fan (top), and digital controllers are usedto regulate and cycle the temperature (bottom). Amplification ismonitored in real time by imaging the array through the transparentplates that make up the top of the device.

FIG. 14, Panels A-B depict the use of a one-color flow-cytometer used todetect PCR amplification products in drops, via fluorescence. Panel A:Schematic of detector, consisting of a 488 nm laser directed into theback of an objective, and focused onto a microfluidic channel throughwhich the droplets flow. The laser excites fluorescent dyes within thedrops, and any emitted light is captured by the objective and imagedonto a photomultiplier tube (PMT) after it is filtered through adichroic mirror and 520±5 nm band pass filter. Panel B: The drops appearas peaks in intensity as a function of time, as shown by the outputvoltage of a PMT, which is proportional to the intensity of the emittedlight, as a function of time for detected fluorescent drops.

FIG. 15, Panels A-C show a schematic of device setup. Panel A: Drops,spacer oil, and 1 M NaCl are introduced to the PDMS device via syringepumps. The picoinjection fluid is introduced using an air pressurecontrol pump. Electrodes from the high voltage amplifier are connectedto a wire submerged in the picoinjection fluid and to the metal needleof the syringe containing the 1 M NaCl “Faraday Mote.” Panel B: Amagnified view of the droplet spacer and picoinjection site. Panel C:Further magnified view of the picoinjection site showing the fluid bulgeat the injection orifice.

FIG. 16, Panels A-B show bright field microscopy images of thepicoinjection site. In the absence of an electric field (Panel A),surfactants prevent coalescence with the injection fluid and a distinctboundary is visible at the droplet/injection fluid interface. When theelectric field is applied, the boundary disappears and reagent isinjected as the droplet passes (Panel B).

FIG. 17, Panels A-C show the volume fraction increase (Vf) of drop sizeafter injection for (Panel A) 100 mM, (Panel B) 50 mM, and (Panel C) 25mM injection fluids. A stronger electric field more readily ruptures theoil/water interfaces allowing injection over a larger length of thepassing droplets, and larger injection volumes. Higher molarities ofdissolved electrolytes produce stronger electric fields at the injectionsite for a given voltage, also increasing injection volume. The errorbars represent 1 standard deviation in either direction for >1200 dropssampled at each point.

FIG. 18 is a heat map showing injection volume as a function of appliedvoltage and the molarity of dissolved NaCl in the injection fluid.Arrows/ticks indicate data points. The injection volume can be adjustedin the range of 0-36 pL with a resolution of ˜2.6 pL 5 (4% Vf) with 100Vincrements of the applied signal. The largest injected volumes were 3000V with the 100 mM fluid. Increasing electric field above this allows forelectrowetting, causing drops to spontaneously form at the picoinjector,adversely affecting injection efficacy and consistency.

FIG. 19 shows ethidium bromide stained 2% agarose gel. Total RNAisolated from an MCF7 human cell line was encapsulated in drops andpicoinjected with an RT-PCR reaction mixture either with (+) or without50 (−) reverse transcriptase (RT) and Taq DNA polymerase. Non-emulsifiedcontrol reactions were performed in parallel. Only reactions receivingenzyme generated the expected 100 bp amplicon. Both positive control andpicoinjected reactions produced PCR products, demonstrating that theelectric field generated during picoinjection is 55 biologicallycompatible with DNA, reverse transcriptase, and Taq.

FIG. 20, Panels A-B show adding reagents via multiple dropletcoalescence. Panel A: A schematic of a microfluidic device for addingreagents via multiple droplet coalescence. The reagent to add isintroduced from below, along with oil, into a very small drop maker.This leads to the production of a train of very small drops at a highfrequency. The drops to which the reagent is to be added are injected,spaced by oil, from the left and then the streams combine where thechannel intersects with the outlet of the tiny drop maker. Because thereagent drops are much smaller than the target drops, they areintroduced at a high rate frequency, and so many (tens or more) of thesedrops are injected for every one target drop. Due to their small sizethey flow faster than the larger drops and collect behind them so that,by the time the reach the electrode channels they are in contact and canbe coalesced by the electric field. Panel B: Close-up of the coalescenceregion in such a microfluidic device. Drops flow from left to the right.A train of tiny droplets form behind the droplet to which they are to beadded. Once the tiny droplets and the droplet pass through thecoalescence region, the electrodes cause the tiny droplets to merge intothe droplet. The resulting output on the right is a droplet thatcontains the reagent(s) that were present in the tiny droplets.

FIG. 21 shows a schematic of a microfluidic device whereby amicrodroplet may be purified. That is, a majority of the fluid in thedrop is replaced it with a purified solution, without removing anydiscrete reagents that may be encapsulated in the drop, such a cells orbeads. The microdroplet is first injected with a solution to dilute anyimpurities within it. The diluted microdroplet is then flowed through amicrofluidic channel on which an electric field is being applied usingelectrodes. Due to the dielectrophoretic forces generated by the field,as the cells or other discrete reagents pass through the field they willbe displaced in the flow. The drops are then split, so that all theobjects end up in one microdroplet. Accordingly, the initialmicrodroplet has been washed, in that the contaminants may be removedwhile the presence and/or concentration of discrete reagents, such asbeads or cells, that may be encapsulated within the droplet aremaintained in the resulting microdroplet.

FIG. 22, Panels A-B show sorting. Droplets enter from the right and flowto the left, passing by the electrodes. The drops are thus sorted on thepresence (Panel A; droplets flow into the top output) or absence of aparticular property (Panel B; droplets flow into the bottom output).

FIG. 23 shows a schematic of a coalescence process, starting with theformation of double emulsions (E2) from a reinjected single emulsion(E1) in a hydrophilic channel (top, left). These are turned into tripleemulsions (E3) at a hydrophobic junction (bottom, left), which are thencoalesced using an electric field into an inverted E2 (E2′, bottom,right).

FIG. 24, Panels A-D show microscope images of (a) double emulsions (E2)formation, (b) triple emulsion (E3) formation, (c) E3 coalescence, and(d) the final inverted E2 (E2′) products. The scale bar applies to allimages.

FIG. 25, Panels A-B show two fast-camera time series showing E3coalescence into E2′. The oil shell of the inner E1 is false-coloredblue.

FIG. 26, Panels A-C show microfluidic devices and digital RT-PCRworkflow used in the study of Example 5. (A) Drops containing RNA andRT-PCR reagents are created with a microfluidic T-junction and carrieroil. Brightfield microscopy images of the drop formation are shownbelow, the middle image showing the generation of one population ofdrops from a single reaction mixture, and the lower the generation oftwo populations from two mixtures. (B) After formation, the drops arepicoinjected with reverse transcriptase using a picoinjection channeltriggered by an electric field, applied by an electrode channelimmediately opposite the picoinjector. (C) The picoinjected drops arecollected into a tube, thermocycled, and imaged with a fluorescentmicroscope.

FIG. 27, Panels A-C show digital RT-PCR TaqMan® assays in microfluidicdrops following picoinjection of reverse transcriptase. (A) ControlRT-PCR reactions containing PC3 cell total RNA were emulsified on aT-junction drop maker, thermocycled, and imaged. FAM (green)fluorescence indicates TaqMan® detection of an EpCAM transcript and Cy5(red) indicates detection of CD44 transcripts. Brightfield images (BF)of the same drops are shown in the image panel on the far right. (B)RT-PCR reactions lacking reverse transcriptase were emulsified on aT-junction drop maker and subsequently picoinjected with reversetranscriptase. Picoinjection fluid is pictured as dark gray in theschematic diagram on the left. Brightfield images demonstrate that thedrops roughly doubled in size after picoinjection. (C) RT-PCR reactionssubjected to picoinjection omitting the reverse transcriptase show noTaqMan® signal for EpCAM and CD44, demonstrating the specificity of theTaqMan® assay. The red arrows indicate the direction of emulsion flow inthe illustrations. Scale bars=100 μm.

FIG. 28, Panels A-B show a comparison of digital RT-PCR detection ratesbetween control drops and drops that were picoinjected with reversetranscriptase. (A) Scatter plots of FAM and Cy5 drop intensities for acontrol sample (left) and picoinjected sample (right). The gatingthresholds used to label a drop as positive or negative for TaqMan®signal are demarcated by the lines, and divide the scatter plot intoquadrants, (−,−), (−,+), (+,−), (+,+). (B) The bar graph shows theaverage TaqMan® positive drop count with picoinjection relative to thenormalized count for CD44 and EpCAM TaqMan® assays for controlpopulations. The data represent the average of four independentexperimental replicates.

FIG. 29, Panels A-B shows that picoinjection enables analysis ofdiscrete drop populations. (A) Non-picoinjected drops. Control RT-PCRreactions containing mixed PC3 cell total RNA and Raji cell total RNAwere emulsified with a T-junction drop maker, thermocycled, and imaged.Merged FAM and HEX fluorescent images are shown with FAM (green)fluorescence indicating TaqMan® detection of an EpCAM transcript and HEX(red) indicating the presence of PTPRC transcripts. The yellow dropsindicate the presence of multiplexed TaqMan® assays, where EpCAM andPTPRC transcripts were co-encapsulated in the same drop. The brightfieldimages (BF) are shown in the panel on the right. (B) Picoinjected drops.A double T-junction drop maker simultaneously created two populations ofdrops that were immediately picoinjected. One drop maker created dropscontaining only Raji cell RNA, and the other drops containing only PC3cell RNA. Both drop types initially lack reverse transcriptase, which isadded via picoinjection just downstream of the drop makers. Theoverwhelming majority of drops display no multiplexing, demonstratingthat transfer of material during picoinjection is very rare. The redarrows indicate the direction of emulsion flow in the illustrations.Scale bars=100 μm.

FIG. 30, Panels A-B shows a dual transcript detection analysis,indicating minimal cross-contamination during picoinjection. (A) Scatterplots of FAM and HEX drop intensities for a co-encapsulated controlsample (left) and dual population picoinjected sample (right). Usingthis analysis, large numbers of TaqMan® multiplexed drops wereidentified in the co-encapsulated controls that were virtually absent inthe dual population picoinjected drops (upper right quadrants of gatedscatter plots). (B) A bar graph of different bright drop populationsrelative to the total bright count for co-encapsulation control and fordual population picoinjection. The data represent the average of threeexperimental replicates.

FIG. 31 Panels A-B shows that dual populations of RNA drops can bestored offline and picoinjected at a later time. (A) An emulsion wasmade consisting of two populations of drops, one containing RNArecovered from Raji cells, and the other from PC3 cells. The drops werecollected into a syringe, incubated off chip, and then re-introducedinto a microfluidic device to picoinject. The drops were then collected,thermocycled, and imaged. These drops are somewhat more polydisperse anddisplayed higher multiplexing rates (1%) than the drops picoinjected onthe same device on which they were formed, which is most likely due tomerger of some of the drops during incubation and reinjection. Theability to reinject emulsions following incubation to add reagents maybe important for numerous droplet-based molecular biology assays. (B)Brightfield images of picoinjected emulsions. Scale bars=100 μm.

FIG. 32 shows an embodiment of a single cell RT-PCR microfluidic deviceas described herein.

FIG. 33 shows the effect of including ridge structures in a microfluidicdevice channel downstream of a droplet forming junction. A T-junctiondrop maker without ridges is shown to the left. As the flow rate ratiois increased, the drop maker stops forming drops and instead forms along jet. This is due to the jet wetting the channel walls and adhering,preventing the formation of drops. On the right, a similar T-junction isshown with ridge structures. The ridges trap a suitable phase, e.g., ahydrophobic oil phase, near the walls, making it difficult for theaqueous phase to wet. This allows the device to form drops at muchhigher flow rate ratios before it eventually wets at R=0.9. This showsthat inclusion of the ridges allows the drop maker to function over amuch wider range than if the ridges are omitted. The channel widths are30 microns and the peaks of the ridges are about 5-10 microns. The topand bottom sets of images correspond to experiments performs withdifferent microfluidic devices.

FIG. 34 provides a flow diagram showing a general fabrication scheme foran embodiment of a liquid electrode as described herein.

FIG. 35 provides a sequence of three images taken at different times asan electrode channel is being filled with salt water (time courseproceeds from left to right; Panels A-C). The salt water is introducedinto the inlet of the channel and pressurized, causing it to slowly fillthe channel. The air that is originally in the channel is pushed intothe PDMS so that, by the end, it is entirely filled with liquid.

FIG. 36 shows electric field lines simulated for various liquidelectrode configurations. The simulations are of positive and groundelectrodes showing equipotential lines for three different geometries.

FIG. 37 provides two images of a droplet merger device that merges largedrops with small drops utilizing liquid electrodes. To merge the drops,an electric field is applied using a salt-water electrode. When thefield is off, no merger occurs (right) and when it is on, the dropsmerge (left).

FIG. 38 provides two different views of a three dimensions schematicshowing a device which may be used to encapsulate single emulsions indouble emulsions. It includes a channel in which the single emulsionsare introduced, which channel opens up into a large channel in whichadditional aqueous phase is added. This focuses the injected dropsthrough an orifice, causing them to be encapsulated in oil drops andforming water-in-oil-in-water double emulsions.

FIG. 39 provides two schematics of PDMS slabs that may be used toconstruct a double emulsification device. The slab on the left haschannels with two heights—short channels for the droplet reinjection andconstriction channels (see previous Figure) and tall channels for theaqueous phase and outlets. The slab on the right has only the tallchannels. To complete the device, the slabs are aligned and sealedtogether so that the channels are facing. The devices are bonded usingplasma oxidation.

FIG. 40 provides a microscope image of a double emulsification deviceencapsulating a reinjected single emulsions in double emulsions. Thereinjected single emulsions enter from above and are encapsulated in theconstriction shown in the center of the device. They then exit as doubleemulsions, four of which are shown towards the bottom of the device.

FIG. 41 provides fluorescent microscope images of fluorescent doubleemulsions. The image on the left shows double emulsions formed byshaking the fluids, which results in a large amount of polydispersityand a small number of drops of the appropriate size for FACS sorting.The image on the right shows double emulsions made with the microfluidicprocess disclosed herein, which are much more monodisperse.

FIG. 42 provides a histogram of the drop areas for shaken vs.device-created double emulsions. The device-created double emulsions aremuch more monodisperse, as demonstrated by the peak.

FIG. 43 shows FACS fluorescence and scattering data for microfluidicdevice generated double emulsions according to the present disclosure.The upper plot shows the intensity histogram of the population asmeasured with the FITC channel (˜520 nm) of the FACS. The plots belowshow the forward and side scattering of the drops, gated according toFITC signal.

FIG. 44 shows FACS fluorescence and scattering data for shaken doubleemulsions. The upper plot shows the intensity histogram of thepopulation as measured with the FITC channel (˜520 nm) of the FACS. Theplots below show the forward and side scattering of the drops, gatedaccording to FITC signal.

FIG. 45 provides a histogram of droplet intensity as read out with theFACS (FITC channel) for four different concentrations of encapsulateddye. The dye is composed of fluorescently-labeled BSA.

FIG. 46 shows the results of an experiment designed to test thedetection rate of the FACS-run drops. Two populations of drops werecreated, one with labeled BSA fluorescent at 520 nm, and another withBSA fluorescent at 647 nm. The two populations were then mixed indefined ratios and the samples were run on FACS. The measured ratio wasfound to agree with the known ratio, demonstrating that the FACSmeasurements are accurate over this range.

FIG. 47 shows emulsions containing three different concentrations ofDNA. All drops contain TaqMan® probes for the DNA target, but the targetis encapsulated at limiting concentration, so that only the drops thatget a target undergo amplification. When the target concentration isreduced, the fraction of fluorescent drops goes down. The lower plotsshow the drops after being encapsulated in double emulsions and screedon FACS.

FIG. 48 shows emulsions containing three concentrations of DNA lowerthan those in the previous Figure. All drops contain TaqMan® probes forthe DNA target, but the target is encapsulated at limitingconcentration, so that only the drops that get a target undergoamplification. When the target concentration is reduced, the fraction offluorescent drops goes down. The lower plots show the drops after beingencapsulated in double emulsions and screed on FACS.

FIG. 49 shows emulsions as for FIGS. 47 and 48 at the lowest DNAconcentration of the three Figures. The lower plot shows the drops afterbeing encapsulated in double emulsions and screed on FACS.

FIG. 50 shows a plot of the detected number of positives by FACSanalysis of double emulsions plotted versus the number of positivesdetected by imaging the drops before double emulsification using afluorescent microscope. The results agree with one another over the twodecades tested.

FIG. 51 provides a plot showing the fraction of drops that are positiveas a function of the log-2 concentration. As the concentration of DNAgoes up, more drops become fluorescent because more of them contain atleast a single molecule.

FIG. 52 provides images showing drops in which a TaqMan® PCR reactionhas been performed with encapsulated Azospira. The upper imagescorrespond to a reaction in which a 110 bp amplicon was produced,whereas the lower images correspond to a 147 bp amplicon.

FIG. 53 shows a picture of a gel showing bands corresponding to theamplicons of two TaqMan® PCR reactions, one for a 464 bp amplicon andone for a 550 bp amplicon.

FIG. 54 shows a picture of a gel validating that PCR reactions can bemultiplexed by adding multiple primer sets to a sample containingbacteria.

FIG. 55 shows results for the PCR amplification of Azospira amplicons(left) and FACS analysis of Azospira containing double emulsions(right).

DETAILED DESCRIPTION

Methods for the detection of components from biological samples areprovided. In certain aspects, the methods may be used to detect and/orquantify specific components in a biological sample, such as tumor cells(e.g., circulating tumor cells). Systems and devices for use inpracticing methods of the invention are also provided.

The subject methods and devices may find use in a wide variety ofapplications, such as the detection of cancer, detection of aneuploidyfrom DNA circulating in a mother's blood stream, monitoring diseaseprogression, analyzing the DNA or RNA content of cells, and a variety ofother applications in which it is desired to detect and/or quantifyspecific components in a biological sample.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, and as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andexemplary methods and materials may now be described. Any and allpublications mentioned herein are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amicrodroplet” includes a plurality of such microdroplets and referenceto “the microdroplet” includes reference to one or more microdroplets,and so forth.

It is further noted that the claims may be drafted to exclude anyelement which may be optional. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.To the extent such publications may set out definitions of a term thatconflict with the explicit or implicit definition of the presentdisclosure, the definition of the present disclosure controls.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Methods

As summarized above, aspects of the invention include methods for thedetection of components from biological samples. Aspects include methodsfor the detection, quantification, and/or genotyping of cells, e.g.normal cells (i.e., non-tumor cells), tumor cells or CTCs.

As used herein, the phrase “biological sample” encompasses a variety ofsample types obtained from an individual and can be used in a diagnosticor monitoring assay. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, cells, serum, plasma, biological fluid, and tissue samples.“Biological sample” includes cells; biological fluids such as blood,cerebrospinal fluid, semen, saliva, and the like; bile; bone marrow;skin (e.g., skin biopsy); and antibodies obtained from an individual.

As described more fully herein, in various aspects the subject methodsmay be used to detect a variety of components from such biologicalsamples. Components of interest include, but are not necessarily limitedto, cells (e.g., circulating cells and/or circulating tumor cells),polynucleotides (e.g., DNA and/or RNA), polypeptides (e.g., peptidesand/or proteins), and many other components that may be present in abiological sample.

“Polynucleotides” or “oligonucleotides” as used herein refer to linearpolymers of nucleotide monomers, and may be used interchangeably.Polynucleotides and oligonucleotides can have any of a variety ofstructural configurations, e.g., be single stranded, double stranded, ora combination of both, as well as having higher order intra- orintermolecular secondary/tertiary structures, e.g., hairpins, loops,triple stranded regions, etc. Polynucleotides typically range in sizefrom a few monomeric units, e.g. 5-40, when they are usually referred toas “oligonucleotides,” to several thousand monomeric units. Whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters (upper or lower case), such as “ATGCCTG,” it will be understoodthat the nucleotides are in 5′→3′ order from left to right and that “A”denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, “I” denotes deoxyinosine, “U”denotes uridine, unless otherwise indicated or obvious from context.Unless otherwise noted the terminology and atom numbering conventionswill follow those disclosed in Strachan and Read, Human MolecularGenetics 2 (Wiley-Liss, New York, 1999).

The terms “polypeptide,” “peptide,” and “protein,” used interchangeablyherein, refer to a polymeric form of amino acids of any length. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxyl group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature, J. Biol. Chem., 243 (1969), 3552-3559 is used.

In certain aspects, methods are provided for counting and/or genotypingcells, including normal cells or tumor cells, such as CTCs. A feature ofsuch methods is the use of microfluidics.

FIG. 1 presents a non-limiting, simplified representation of one type ofa microfluidics system and method of the present disclosure. Theparticular application depicted in FIG. 1 is the detection and/orgenotyping of cells, e.g., tumor cells, from a biological sample. In onesuch method, nucleated blood cells may be obtained from a biologicalsample from a subject. The nucleated blood cells are encapsulated intoindividual drops using an encapsulation device (left). The drops maythen be injected with a lysis buffer and incubated at conditions thataccelerate cell lysis (e.g., at 37° C.). The drops may be injected witha PCR mix that includes one or more primers targeting characteristiconcogenic mutations (center). The drops containing the PCR mix may beflowed through a channel that incubates the droplets under conditionseffective for PCR. In the figure, this is achieved by flowing the dropsthrough a channel that snakes over various zones maintained at 65° C.and 95° C. As the drops move through the zones, their temperaturecycles, as needed for PCR. During the PCR reaction, if a dropletcontains a genome of a cell with a mutation for which the primer(s) aredesigned to detect, amplification is initiated. The presence of theseparticular PCR products may be detected by, for example, a fluorescentoutput that turns the drop fluorescent (FIGS. 3-4). The drops may thusbe scanned, such as by using flow cytometry, to detect the presence offluorescent drops (FIG. 14, Panels A-B). In certain aspects, the dropsmay also be sorted using, for example, droplet sorting to recover dropsof interest (right). Using the nomenclature of the current disclosure,the steps described above are thus performed “under microfluidiccontrol.” That is, the steps are performed on one or more microfluidicsdevices.

FIG. 2, Panels A-E depict a microfluidics system involving many of thegeneral principles and steps described above. Here, yeast cells (blackspecks) enter from the far left and are encapsulated into drops, shownat low (4× objective; Panel A) and high magnification (10× objective;Panel B). The drops are incubated to allowing the yeast to secrete theirproduct (Panel C); this produces a fluorescent compound in the drops, sothat drops containing efficient producers quickly become fluorescent(Panel D). The drops are then sorted to extract the most efficient yeastusing a microfluidic sorter (Panel E).

Encapsulating a component from a biological sample may be achieved byany convenient means. FIG. 5 presents but one possible example, in whichdroplets are formed in a massively parallel fashion a serial bisectiondevice. For instance, cell-containing solution may be injected from theleft and formed into large drops, which flow into the serial bisectionarray and are split into small drops; drops shown to the far right are25 mm in diameter. Encapsulation approaches of interest also include,but are not limited to, hydrodynamically-triggered drop formation andthose described by Link, et al., Phys. Rev. Lett. 92, 054503 (2004), thedisclosure of which is incorporated herein by reference.

As evidenced by FIGS. 1, 4, and 6, a feature of certain methods of thepresent disclosure is the use of a polymerase chain reaction (PCR)-basedassay to detect the presence of certain oligonucleotides and/oroncogene(s) present in cells. Examples of PCR-based assays of interestinclude, but are not limited to, quantitative PCR (qPCR), quantitativefluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), real timePCR (RT-PCR), single cell PCR, PCR-RFLP/RT-PCR-RFLP, hot start PCR,nested PCR, in situ polony PCR, in situ rolling circle amplification(RCA), bridge PCR, picotiter PCR and emulsion PCR. Other suitableamplification methods include the ligase chain reaction (LCR),transcription amplification, self-sustained sequence replication,selective amplification of target polynucleotide sequences, consensussequence primed polymerase chain reaction (CP-PCR), arbitrarily primedpolymerase chain reaction (AP-PCR), degenerate oligonucleotide-primedPCR (DOP-PCR) and nucleic acid based sequence amplification (NABSA).

A PCR-based assay may be used to detect the presence of certain gene(s),such as certain oncogene(s). FIG. 4, Panels A-B depict a PCR-based assayto detect oncogenes. In this assay, one or more primers specific to eachoncogene of interest are reacted with the genome of each cell. Theseprimers have sequences specific to the particular oncogene, so that theywill only hybridize and initiate PCR when they are complimentary to thegenome of the cell. If an oncogene is present and the primer is a match,large many copies of the oncogene are created. To determine whether anoncogene is present, the PCR products may be detected through an assayprobing the liquid of the drop, such as by staining the solution with anintercalating dye, like SybrGreen or ethidium bromide, hybridizing thePCR products to a solid substrate, such as a bead (e.g., magnetic orfluorescent beads, such as Luminex beads), or detecting them through anintermolecular reaction, such as FRET. These dyes, beads, and the likeare each examples of a “detection component,” a term that is usedbroadly and generically herein to refer to any component that is used todetect the presence or absence of PCR product(s).

A great number of variations of these basic approaches will now beoutlined in greater detail below.

Detecting Rare Cells (e.g., Tumor Cells)

Aspects of the subject methods involve detecting the presence of one ormore subset of cells (e.g., tumor cells) in a biological sample. Such ascheme is depicted in FIG. 6. To use this approach for the detection oftumor cells, a biological sample (e.g., whole blood) may be recoveredfrom a subject using any convenient means. The biological sample may beprocessed to remove components other than cells using, for example,processing steps such as centrifugation, filtration, and the like.

Each cell in the biological sample is then encapsulated into a dropletusing a microfluidic device, such as that shown in FIGS. 5 and/or 8.Using the example from FIG. 5, the cell-containing solution is injectedfrom the left and formed into large drops, which flow into the serialbisection array and are split into smaller droplets. Other methods ofencapsulating cells into droplets are known in the art. Where desired,the cells may be stained with one or more antibodies and/or probes priorto encapsulating them into drops. As used herein, the terms “drop,”“droplet,” and “microdroplet” may be used interchangeably, to refer totiny spheres containing an aqueous phase, e.g., water, generally rangingfrom 0.1 to 1000 μm in diameter, which may be used to encapsulate cells,DNA, enzymes, and other components. Accordingly, the terms may be usedto refer to a droplet produced in, on, or by a microfluidics device.

One or more lysing agents may also be added to the droplets containing acell, under conditions in which the cell(s) may be caused to burst,thereby releasing their genomes. The lysing agents may be added afterthe cells are encapsulated into microdroplets. Any convenient lysingagent may be employed, such as proteinase K or cytotoxins. In particularembodiments, cells may be co-encapsulated in drops with lysis buffercontaining detergents such as Triton X100 and/or proteinase K. Thespecific conditions in which the cell(s) may be caused to burst willvary depending on the specific lysing agent used. For example, ifproteinase K is incorporated as a lysing agent, the microdroplets may beheated to about 37-60° C. for about 20 min to lyse the cells and toallow the proteinase K to digest cellular proteins, after which they maybe heated to about 95° C. for about 5-10 min to deactivate theproteinase K.

In certain aspects, cell lysis may also, or instead, rely on techniquesthat do not involve addition of lysing agent. For example, lysis may beachieved by mechanical techniques that may employ various geometricfeatures to effect piercing, shearing, abrading, etc. of cells. Othertypes of mechanical breakage such as acoustic techniques may also beused. Further, thermal energy can also be used to lyse cells. Anyconvenient means of effecting cell lysis may be employed in the methodsdescribed herein.

Primers may be introduced into the droplet, for each of the genes, e.g.,oncogenes, to be detected. Hence, in certain aspects, primers for alloncogenes may be present in the droplet at the same time, therebyproviding a multiplexed assay. The droplets are temperature-cycled sothat droplets containing cancerous cells, for example, will undergo PCR.During this time, only the primers corresponding to oncogenes present inthe genome will induce amplification, creating many copies of theseoncogenes in the droplet. Detecting the presence of these PCR productsmay be achieved by a variety of ways, such as by using FRET, stainingwith an intercalating dye, or attaching them to a bead. For moreinformation on the different options for this, see the sectiondescribing variations of the technique. The droplet may be opticallyprobed to detect the PCR products (FIG. 14). Optically probing thedroplet may involve counting the number of tumor cells present in theinitial population, and/or to allow for the identification the oncogenespresent in each tumor cell.

The subject methods may be used to determine whether a biological samplecontains particular cells of interest, e.g., tumor cells, or not. Incertain aspects, the subject methods may include quantifying the numberof cells of interest, e.g., tumor cells, present in a biological sample.Quantifying the number of cells of interest, e.g., tumor cells, presentin a biological sample may be based at least in part on the number ofmicrodroplets in which PCR amplification products were detected. Forexample, microdroplets may be produced under conditions in which themajority of droplets are expected to contain zero or one cells. Thosedroplets that do not contain any cells may be removed, using techniquesdescribed more fully herein. After performing the PCR steps outlinedabove, the total number of microdroplets that are detected to containPCR products may be counted, so as to quantify the number of cells ofinterest, e.g., tumor cells, in the biological sample. In certainaspects, the methods may also include counting the total number ofmicrodroplets, so as to determine the fraction or percentage of cellsfrom the biological sample that are cells of interest, e.g., tumorcells.

PCR

As summarized above, in practicing methods of the invention a PCR-basedassay may be used to detect the presence of certain genes of interest,e.g., oncogene(s), present in cells. The conditions of such PCR-basedassays may vary in one or more ways.

For instance, the number of PCR primers that may be added to amicrodroplet may vary. The term “primer” may refer to more than oneprimer and refers to an oligonucleotide, whether occurring naturally, asin a purified restriction digest, or produced synthetically, which iscapable of acting as a point of initiation of synthesis along acomplementary strand when placed under conditions in which synthesis ofa primer extension product which is complementary to a nucleic acidstrand is catalyzed. Such conditions include the presence of fourdifferent deoxyribonucleoside triphosphates and apolymerization-inducing agent such as DNA polymerase or reversetranscriptase, in a suitable buffer (“buffer” includes substituentswhich are cofactors, or which affect pH, ionic strength, etc.), and at asuitable temperature. The primer is preferably single-stranded formaximum efficiency in amplification.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Complementarity need not be perfect;stable duplexes may contain mismatched base pairs or unmatched bases.Those skilled in the art of nucleic acid technology can determine duplexstability empirically considering a number of variables including, forexample, the length of the oligonucleotide, percent concentration ofcytosine and guanine bases in the oligonucleotide, ionic strength, andincidence of mismatched base pairs.

The number of PCR primers that may be added to a microdroplet may rangefrom about 1 to about 500 or more, e.g., about 2 to 100 primers, about 2to 10 primers, about 10 to 20 primers, about 20 to 30 primers, about 30to 40 primers, about 40 to 50 primers, about 50 to 60 primers, about 60to 70 primers, about 70 to 80 primers, about 80 to 90 primers, about 90to 100 primers, about 100 to 150 primers, about 150 to 200 primers,about 200 to 250 primers, about 250 to 300 primers, about 300 to 350primers, about 350 to 400 primers, about 400 to 450 primers, about 450to 500 primers, or about 500 primers or more.

These primers may contain primers for one or more gene of interest, e.g.oncogenes. The number of primers for genes of interest that are addedmay be from about one to 500, e.g., about 1 to 10 primers, about 10 to20 primers, about 20 to 30 primers, about 30 to 40 primers, about 40 to50 primers, about 50 to 60 primers, about 60 to 70 primers, about 70 to80 primers, about 80 to 90 primers, about 90 to 100 primers, about 100to 150 primers, about 150 to 200 primers, about 200 to 250 primers,about 250 to 300 primers, about 300 to 350 primers, about 350 to 400primers, about 400 to 450 primers, about 450 to 500 primers, or about500 primers or more. Genes and oncogenes of interest include, but arenot limited to, BAX, BCL2L1, CASP8, CDK4, ELK1, ETS1, HGF, JAK2, JUNB,JUND, KIT, KITLG, MCL1, MET, MOS, MYB, NFKBIA, EGFR, Myc, EpCAM, NRAS,PIK3CA, PML, PRKCA, RAF1, RARA, REL, ROS1, RUNX1, SRC, STAT3, CD45,cytokeratins, CEA, CD133, HER2, CD44, CD49f, CD146, MUC1/2, and ZHX2.

Such primers and/or reagents may be added to a microdroplet in one step,or in more than one step. For instance, the primers may be added in twoor more steps, three or more steps, four or more steps, or five or moresteps. Regardless of whether the primers are added in one step or inmore than one step, they may be added after the addition of a lysingagent, prior to the addition of a lysing agent, or concomitantly withthe addition of a lysing agent. When added before or after the additionof a lysing agent, the PCR primers may be added in a separate step fromthe addition of a lysing agent.

Once primers have been added to a microdroplet, the microdroplet may beincubated under conditions allowing for PCR. The microdroplet may beincubated on the same microfluidic device as was used to add theprimer(s), or may be incubated on a separate device. In certainembodiments, incubating the microdroplet under conditions allowing forPCR amplification is performed on the same microfluidic device used toencapsulate the cells and lyse the cells. Incubating the microdropletsmay take a variety of forms. In certain aspects, the drops containingthe PCR mix may be flowed through a channel that incubates the dropletsunder conditions effective for PCR. Flowing the microdroplets through achannel may involve a channel that snakes over various temperature zonesmaintained at temperatures effective for PCR. Such channels may, forexample, cycle over two or more temperature zones, wherein at least onezone is maintained at about 65° C. and at least one zone is maintainedat about 95° C. As the drops move through such zones, their temperaturecycles, as needed for PCR. The precise number of zones, and therespective temperature of each zone, may be readily determined by thoseof skill in the art to achieve the desired PCR amplification.

In other embodiments, incubating the microdroplets may involve the useof a device of the general types depicted in FIG. 12, Panels A-C, andFIG. 13; a device of this general type may be referred to herein as a“Megadroplet Array.” In such a device, an array of hundreds, thousands,or millions of traps indented into a channel (e.g., a PDMS channel) sitabove a thermal system (FIG. 12, Panel A). The channel may bepressurized, thereby preventing gas from escaping. The height of themicrofluidic channel is smaller than the diameter of the drops, causingdrops to adopt a flattened pancake shape. When a drop flows over anunoccupied indentation, it adopts a lower, more energetically favorable,radius of curvature, leading to a force that pulls the drop entirelyinto the trap (FIG. 12, Panel B). By flowing drops as a close pack, itis ensured that all traps on the array are occupied, as illustrated inFIG. 12, Panel C. The entire device may be thermal cycled using aheater.

In certain aspects, the heater includes a Peltier plate, heat sink, andcontrol computer. The Peltier plate allows for the heating or cooling ofthe chip above or below room temperature by controlling the appliedcurrent. To ensure controlled and reproducible temperature, a computermay monitor the temperature of the array using integrated temperatureprobes, and may adjust the applied current to heat and cool as needed. Ametallic (e.g. copper) plate allows for uniform application of heat anddissipation of excess heat during cooling cycles, enabling cooling fromabout 95° C. to about 60° C. in under about one minute.

Methods of the invention may also include introducing one or more probesto the microdroplet. As used herein with respect to nucleic acids, theterm “probe” refers to a labeled oligonucleotide which forms a duplexstructure with a sequence in the target nucleic acid, due tocomplementarity of at least one sequence in the probe with a sequence inthe target region. The probe, preferably, does not contain a sequencecomplementary to sequence(s) used to prime the polymerase chainreaction. The number of probes that are added may be from about one to500, e.g., about 1 to 10 probes, about 10 to 20 probes, about 20 to 30probes, about 30 to 40 probes, about 40 to 50 probes, about 50 to 60probes, about 60 to 70 probes, about 70 to 80 probes, about 80 to 90probes, about 90 to 100 probes, about 100 to 150 probes, about 150 to200 probes, about 200 to 250 probes, about 250 to 300 probes, about 300to 350 probes, about 350 to 400 probes, about 400 to 450 probes, about450 to 500 probes, or about 500 probes or more. The probe(s) may beintroduced into the microdroplet prior to, subsequent with, or after theaddition of the one or more primer(s). Probes of interest include, butare not limited to, TaqMan® probes (e.g., as described in Holland, P.M.; Abramson, R. D.; Watson, R.; Gelfand, D. H. (1991). “Detection ofspecific polymerase chain reaction product by utilizing the 5′-3′exonuclease activity of Thermus aquaticus DNA polymerase”. PNAS, 88(16): 7276-7280).

In certain embodiments, an RT-PCR based assay may be used to detect thepresence of certain transcripts of interest, e.g., oncogene(s), presentin cells. In such embodiments, reverse transcriptase and any otherreagents necessary for cDNA synthesis are added to the microdroplet inaddition to the reagents used to carry out PCR described herein(collectively referred to as the “RT-PCR reagents”). The RT-PCR reagentsare added to the microdroplet using any of the methods described herein.Once reagents for RT-PCR have been added to a microdroplet, themicrodroplet may be incubated under conditions allowing for reversetranscription followed by conditions allowing for PCR as describedherein. The microdroplet may be incubated on the same microfluidicdevice as was used to add the RT-PCR reagents, or may be incubated on aseparate device. In certain embodiments, incubating the microdropletunder conditions allowing for RT-PCR is performed on the samemicrofluidic device used to encapsulate the cells and lyse the cells.

In certain embodiments, the reagents added to the microdroplet forRT-PCR or PCR further includes a fluorescent DNA probe capable ofdetecting real-time RT-PCR or PCR products. Any suitable fluorescent DNAprobe can be used including, but not limited to SYBR Green, TaqMan®,Molecular Beacons and Scorpion probes. In certain embodiments, thereagents added to the microdroplet include more than one DNA probe,e.g., two fluorescent DNA probes, three fluorescent DNA probes, or fourfluorescent DNA probes. The use of multiple fluorescent DNA probesallows for the concurrent measurement of RT-PCR or PCR products in asingle reaction.

Double PCR

To amplify rare transcripts, a microdroplet that has undergone afirst-step RT-PCR or PCR reaction as described herein may be furthersubjected to a second step PCR reaction. In some embodiments, a portionof a microdroplet that has undergone a first-step RT-PCR or PCR reactionis extracted from the microdroplet and coalesced with a dropletcontaining additional PCR reagents, including, but not limited toenzymes (e.g. DNA polymerase), DNA probes (e.g. fluorescent DNA probes)and primers. In certain embodiments, the droplet containing theadditional PCR reagents is larger than the microdroplet that hasundergone the first step RT-PCR or PCR reaction. This may be beneficial,for example, because it allows for the dilution of cellular componentsthat may be inhibitory to the second step PCR. The second step PCRreaction may be carried out on the same microfluidic device used tocarry out the first-step reaction or on a different microfluidic device.

In some embodiments, the primers used in the second step PCR reactionare the same primers used in the first step RT-PCR or PCR reaction. Inother embodiments, the primers used in the second step PCR reaction aredifferent than the primers used in the first step reaction.

Multiplexing

In certain embodiments of the subject methods, multiple biomarkers maybe detected and analyzed for a particular cell. Biomarkers detected mayinclude, but are not limited to, one or more proteins, transcriptsand/or genetic signatures in the cell's genome or combinations thereof.With standard fluorescence based detection, the number of biomarkersthat can be simultaneously interrogated may be limited to the number offluorescent dyes that can be independently visualized within eachmicrodrop. In certain embodiments, the number of biomarkers that can beindividually detected within a particular microdroplet can be increased.For example, this may be accomplished by segregation of dyes todifferent parts of the microdroplet. In particular embodiments, beads(e.g. LUMINEX® beads) conjugated with dyes and probes (e.g., nucleicacid or antibody probes) may be encapsulated in the microdroplet toincrease the number of biomarkers analyzed. In another embodiment,fluorescence polarization may be used to achieve a greater number ofdetectable signals for different biomarkers for a single cell. Forexample, fluorescent dyes may be attached to various probes and themicrodroplet may be visualized under different polarization conditions.In this way, the same colored dye can be utilized to provide a signalfor different probe targets for a single cell. The use of fixed and/orpermeabilized cells (as discussed in greater detail below) also allowsfor increased levels of multiplexing. For example, labeled antibodiesmay be used to target protein targets localized to cellular componentswhile labeled PCR and/or RT-PCR products are free within a microdroplet.This allows for dyes of the same color to be used for antibodies and foramplicons produced by RT-PCR.

Types of Microdroplets

In practicing the methods of the present invention, the composition andnature of the microdroplets may vary. For instance, in certain aspects,a surfactant may be used to stabilize the microdroplets. Accordingly, amicrodroplet may involve a surfactant stabilized emulsion. Anyconvenient surfactant that allows for the desired reactions to beperformed in the drops may be used. In other aspects, a microdroplet isnot stabilized by surfactants or particles.

The surfactant used depends on a number of factors such as the oil andaqueous phases (or other suitable immiscible phases, e.g., any suitablehydrophobic and hydrophilic phases) used for the emulsions. For example,when using aqueous droplets in a fluorocarbon oil, the surfactant mayhave a hydrophilic block (PEG-PPO) and a hydrophobic fluorinated block(Krytox FSH). If, however, the oil was switched to be a hydrocarbon oil,for example, the surfactant would instead be chosen so that it had ahydrophobic hydrocarbon block, like the surfactant ABIL EM90. Inselecting a surfactant, desirable properties that may be considered inchoosing the surfactant may include one or more of the following: (1)the surfactant has low viscosity; (2) the surfactant is immiscible withthe polymer used to construct the device, and thus it doesn't swell thedevice; (3) biocompatibility; (4) the assay reagents are not soluble inthe surfactant; (5) the surfactant exhibits favorable gas solubility, inthat it allows gases to come in and out; (6) the surfactant has aboiling point higher than the temperature used for PCR (e.g., 95 C); (7)the emulsion stability; (8) that the surfactant stabilizes drops of thedesired size; (9) that the surfactant is soluble in the carrier phaseand not in the droplet phase; (10) that the surfactant has limitedfluorescence properties; and (11) that the surfactant remains soluble inthe carrier phase over a range of temperatures.

Other surfactants can also be envisioned, including ionic surfactants.Other additives can also be included in the oil to stabilize the drops,including polymers that increase droplet stability at temperatures above35° C.

Adding Reagents to Microdroplets

In practicing the subject methods, a number of reagents may need to beadded to the microdroplets, in one or more steps (e.g., about 2, about3, about 4, or about 5 or more steps). The means of adding reagents tothe microdroplets may vary in a number of ways. Approaches of interestinclude, but are not limited to, those described by Ahn, et al., Appl.Phys. Lett. 88, 264105 (2006); Priest, et al., Appl. Phys. Lett. 89,134101 (2006); Abate, et al., PNAS, Nov. 9, 2010 vol. 107 no. 4519163-19166; and Song, et al., Anal. Chem., 2006, 78 (14), pp 4839-4849;the disclosures of which are incorporated herein by reference.

For instance, a reagent may be added to a microdroplet by a methodinvolving merging a microdroplet with a second microdroplet thatcontains the reagent(s). The reagent(s) that are contained in the secondmicrodroplet may be added by any convenient means, specificallyincluding those described herein. This droplet may be merged with thefirst microdroplet to create a microdroplet that includes the contentsof both the first microdroplet and the second microdroplet.

One or more reagents may also, or instead, be added using techniquessuch as droplet coalescence, or picoinjection. In droplet coalescence, atarget drop (i.e., the microdroplet) may be flowed alongside amicrodroplet containing the reagent(s) to be added to the microdroplet.The two microdroplets may be flowed such that they are in contact witheach other, but not touching other microdroplets. These drops may thenbe passed through electrodes or other means of applying an electricalfield, wherein the electric field may destabilize the microdroplets suchthat they are merged together.

Reagents may also, or instead, be added using picoinjection. In thisapproach, a target drop (i.e., the microdroplet) may be flowed past achannel containing the reagent(s) to be added, wherein the reagent(s)are at an elevated pressure. Due to the presence of the surfactants,however, in the absence of an electric field, the microdroplet will flowpast without being injected, because surfactants coating themicrodroplet may prevent the fluid(s) from entering. However, if anelectric field is applied to the microdroplet as it passes the injector,fluid containing the reagent(s) will be injected into the microdroplet.The amount of reagent added to the microdroplet may be controlled byseveral different parameters, such as by adjusting the injectionpressure and the velocity of the flowing drops, by switching theelectric field on and off, and the like.

In other aspects, one or more reagents may also, or instead, be added toa microdroplet by a method that does not rely on merging two dropletstogether or on injecting liquid into a drop. Rather, one or morereagents may be added to a microdroplet by a method involving the stepsof emulsifying a reagent into a stream of very small drops, and mergingthese small drops with a target microdroplet (FIG. 20, Panels A-B). Suchmethods shall be referred to herein as “reagent addition throughmultiple-drop coalescence.” These methods take advantage of the factthat due to the small size of the drops to be added compared to that ofthe target drops, the small drops will flow faster than the target dropsand collect behind them. The collection can then be merged by, forexample, applying an electric field. This approach can also, or instead,be used to add multiple reagents to a microdroplet by using severalco-flowing streams of small drops of different fluids. To enableeffective merger of the tiny and target drops, it is important to makethe tiny drops smaller than the channel containing the target drops, andalso to make the distance between the channel injecting the target dropsfrom the electrodes applying the electric field sufficiently long so asto give the tiny drops time to “catch up” to the target drops. If thischannel is too short, not all tiny drops will merge with the target dropand adding less reagent than desired. To a certain degree, this can becompensated for by increasing the magnitude of the electric field, whichtends to allow drops that are farther apart to merge. In addition tomaking the tiny drops on the same microfluidic device, as is shown inFIG. 20, Panels A-B, they can also, or instead, be made offline usinganother microfluidic drop maker or through homogenization and theninjecting them into the device containing the target drops.

Accordingly, in certain aspects a reagent is added to a microdroplet bya method involving emulsifying the reagent into a stream of droplets,wherein the droplets are smaller than the size of the microdroplet;flowing the droplets together with the microdroplet; and merging adroplet with the microdroplet. The diameter of the droplets contained inthe stream of droplets may vary ranging from about 75% or less than thatof the diameter of the microdroplet, e.g., the diameter of the flowingdroplets is about 75% or less than that of the diameter of themicrodroplet, about 50% or less than that of the diameter of themicrodroplet, about 25% or less than that of the diameter of themicrodroplet, about 15% or less than that of the diameter of themicrodroplet, about 10% or less than that of the diameter of themicrodroplet, about 5% or less than that of the diameter of themicrodroplet, or about 2% or less than that of the diameter of themicrodroplet. In certain aspects, a plurality of flowing droplets may bemerged with the microdroplet, such as 2 or more droplets, 3 or more, 4or more, or 5 or more. Such merging may be achieved by any convenientmeans, including but not limited to by applying an electric field,wherein the electric field is effective to merge the flowing dropletwith the microdroplet.

As a variation of the above-described methods, the fluids may bejetting. That is, rather than emulsifying the fluid to be added intoflowing droplets, a long jet of this fluid can be formed and flowedalongside the target microdroplet. These two fluids can then be mergedby, for example, applying an electric field. The result is a jet withbulges where the microdroplets are, which may naturally break apart intomicrodroplets of roughly the size of the target microdroplets before themerger, due to the Rayleigh plateau instability. A number of variantsare contemplated. For instance, one or more agents may be added to thejetting fluid to make it easier to jet, such as gelling agents and/orsurfactants. Moreover, the viscosity of the continuous fluid could alsobe adjusted to enable jetting, such as that described by Utada, et al.,Phys. Rev. Lett. 99, 094502 (2007), the disclosure of which isincorporated herein by reference.

In other aspects, one or more reagents may be added using a method thatuses the injection fluid itself as an electrode, by exploiting dissolvedelectrolytes in solution (FIGS. 15-19). Methods of this general type aredescribed more fully herein in Example 3.

In another aspect, a reagent is added to a drop (e.g., a microdroplet)formed at an earlier time by enveloping the drop to which the reagent isbe added (i.e., the “target drop”) inside a drop containing the reagentto be added (the “target reagent”). In certain embodiments such a methodis carried out by first encapsulating the target drop in a shell of asuitable hydrophobic phase, e.g., oil, to form a double emulsion. Thedouble emulsion is then encapsulated by a drop containing the targetreagent to form a triple emulsion. To combine the target drop with thedrop containing the target reagent, the double emulsion is then burstopen using any suitable method, including, but not limited to, applyingan electric field, adding chemicals that destabilizes the dropletinterface, flowing the triple emulsion through constrictions and othermicrofluidic geometries, applying mechanical agitation or ultrasound,increasing or reducing temperature, or by encapsulating magneticparticles in the drops that can rupture the double emulsion interfacewhen pulled by a magnetic field. Methods of making a triple emulsion andcombining a target drop with a target reagent are described in Example 4provided herein.

Detecting PCR Products

In practicing the subject methods, the manner in which PCR products maybe detected may vary. For example, if the goal is simply to count thenumber of a particular cell type, e.g., tumor cells, present in apopulation, this may be achieved by using a simple binary assay in whichSybrGreen, or any other stain and/or intercalating stain, is added toeach microdroplet so that in the event a characterizing gene, e.g., anoncogene, is present and PCR products are produced, the drop will becomefluorescent. The change in fluorescence may be due to fluorescencepolarization. The detection component may include the use of anintercalating stain (e.g., SybrGreen).

A variety of different detection components may be used in practicingthe subject methods, including using fluorescent dyes known in the art.Fluorescent dyes may typically be divided into families, such asfluorescein and its derivatives; rhodamine and its derivatives; cyanineand its derivatives; coumarin and its derivatives; Cascade Blue and itsderivatives; Lucifer Yellow and its derivatives; BODIPY and itsderivatives; and the like. Exemplary fluorophores includeindocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5,Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, AlexaFluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluoresceinisothiocyanate (FITC), carboxy-fluorescein (FAM), phycoerythrin,rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine(TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen,RiboGreen, and the like. Descriptions of fluorophores and their use, canbe found in, among other places, R. Haugland, Handbook of FluorescentProbes and Research Products, 9th ed. (2002), Molecular Probes, Eugene,Oreg.; M. Schena, Microarray Analysis (2003), John Wiley & Sons,Hoboken, N.J.; Synthetic Medicinal Chemistry 2003/2004 Catalog, Berryand Associates, Ann Arbor, Mich.; G. Hermanson, Bioconjugate Techniques,Academic Press (1996); and Glen Research 2002 Catalog, Sterling, Va.

FIG. 14, Panels A-B depict the use of a one-color flow-cytometer, whichcan be used, for example, to detect tumor cell containing drops. Panel Apresents a schematic of a detector, consisting of a 488 nm laserdirected into the back of an objective, and focused onto a microfluidicchannel through which the droplets flow. The laser may excitefluorescent dyes within the drops, and any emitted light is captured bythe objective and imaged onto a PMT after it is filtered through adichroic mirror and 520±5 nm band pass filter. Turning to Panel B, dropsappear as peaks in intensity as a function of time, as shown by theoutput voltage of a PMT, which is proportional to the intensity of theemitted light, as a function of time for detected fluorescent drops.

FIGS. 3 and 4, Panels A-B further illustrate such a concept. FIG. 3, forexample, is a non-limiting example that depicts digital detection ofBRAF using TaqMan® PCR assays in arrayed microdrops. Fluorescent dropsindicate amplification of the BRAF gene from human genomic DNA, whilenon-fluorescent drops were devoid of the gene. Turning to FIG. 4, PanelsA-B, this scheme is generalized. In FIG. 4, Panel A, a schematic ispresented showing forward and reverse primers being encapsulated in themicrodroplets that target an oncogenic sequence. If the oncogenicsequence is present, the PCR reaction produces double-stranded PCRproducts (Panel A, upper), whereas, if it is not, no products areproduced (Panel A, lower). SybrGreen, or any other type of intercalatingstain, is also present in the drop. The results are depicted by theimages in FIG. 4, Panel B, in that if double-stranded products areproduced, the dye intercalates into them, becoming fluorescent, andturning the drop fluorescent (FIG. 4, Panel B, upper); by contrast, ifno double-stranded products are produced, the dye remainsnon-fluorescent, producing a dim drop (FIG. 4, Panel B, lower).

In other aspects, particularly if a goal is to further characterize theoncogenes present, additional testing may be needed. For instance, inthe case of the multiplex assays described more fully herein (Example2), this may be achieved by having optical outputs that relate which ofthe gene(s) amplified in the drop. An alternative approach would be touse a binary output, for example, with an intercalated stain, to simplydetermine which droplets have any oncogenes. These can then be sorted torecover these drops so that they could be analyzed in greater detail todetermine which oncogenes they contain. To determine the oncogenespresent in such a drop, microfluidic techniques or nonmicrofluidictechniques could be used. Using non-microfluidic techniques, a dropletidentified as containing an oncogene can be placed into a well on awellplate where will be diluted into a larger volume, releasing all ofthe PCR products that were created during the multiplexed PCR reaction.Samples from this well can then be transferred into other wells, intoeach of which would be added primers for one of the oncogenes. Thesewells would then be temperature-cycled to initiate PCR, at which pointan intercalating stain would be added to cause wells that have matchingoncogenes and primers to light up.

In practicing the subject methods, therefore, a component may bedetected based upon, for example, a change in fluorescence. In certainaspects, the change in fluorescence is due to fluorescence resonanceenergy transfer (FRET). In this approach, a special set of primers maybe used in which the 5′ primer has a quencher dye and the 3′ primer hasa fluorescent dye. These dyes can be arranged anywhere on the primers,either on the ends or in the middles. Because the primers arecomplementary, they will exist as duplexes in solution, so that theemission of the fluorescent dye will be quenched by the quencher dye,since they will be in close proximity to one another, causing thesolution to appear dark. After PCR, these primers will be incorporatedinto the long PCR products, and will therefore be far apart from oneanother. This will allow the fluorescent dye to emit light, causing thesolution to become fluorescent. Hence, to detect if a particularoncogene is present, one may measure the intensity of the droplet at thewavelength of the fluorescent dye. To detect if different oncogenes arepresent, this would be done with different colored dyes for thedifferent primers. This would cause the droplet to become fluorescent atall wavelengths corresponding to the primers of the oncogenes present inthe cell.

Sorting

In practicing the methods of the present disclosure, one or more sortingsteps may be employed. Sorting approaches of interest include, by arenot necessarily limited to, approaches that involve the use of membranevalves, bifurcating channels, surface acoustic waves, and/ordielectrophoresis. Sorting approaches of interest further include thosedepicted in FIGS. 2 and 22, Panels A-B, and those described by Agresti,et al., PNAS vol. 107, no 9, 4004-4009; the disclosure of which isincorporated herein by reference. A population may be enriched bysorting, in that a population containing a mix of members having or nothaving a desired property may be enriched by removing those members thatdo not have the desired property, thereby producing an enrichedpopulation having the desired property.

Sorting may be applied before or after any of the steps describedherein. Moreover, two or more sorting steps may be applied to apopulation of microdroplets, e.g., about 2 or more sorting steps, about3 or more, about 4 or more, or about 5 or more, etc. When a plurality ofsorting steps is applied, the steps may be substantially identical ordifferent in one or more ways (e.g., sorting based upon a differentproperty, sorting using a different technique, and the like).

Moreover, droplets may be purified prior to, or after, any sorting step.FIG. 21 presents a schematic of a microfluidic device whereby amicrodroplet may be purified. That is, a majority of the fluid in thedrop is replaced it with a purified solution, without removing anydiscrete reagents that may be encapsulated in the drop, such a cells orbeads. The microdroplet is first injected with a solution to dilute anyimpurities within it. The diluted microdroplet is then flowed through amicrofluidic channel on which an electric field is being applied usingelectrodes. Due to the dielectrophoretic forces generated by the field,as the cells or other discrete reagents pass through the field they willbe displaced in the flow. The drops are then split, so that all theobjects end up in one microdroplet. Accordingly, the initialmicrodroplet has been purified, in that the contaminants may be removedwhile the presence and/or concentration of discrete reagents, such asbeads or cells, that may be encapsulated within the droplet aremaintained in the resulting microdroplet.

Microdroplets may be sorted based on one or more properties. Propertiesof interest include, but are not limited to, the size, viscosity, mass,buoyancy, surface tension, electrical conductivity, charge, magnetism,and/or presence or absence of one or more components. In certainaspects, sorting may be based at least in part upon the presence orabsence of a cell in the microdroplet. In certain aspects, sorting maybe based at least in part based upon the detection of the presence orabsence of PCR amplification products.

Microdroplet sorting may be employed, for example, to removemicrodroplets in which no cells are present. Encapsulation may result inone or more microdroplets, including a majority of the microdroplets, inwhich no cell is present. If such empty drops were left in the system,they would be processed as any other drop, during which reagents andtime would be wasted. To achieve the highest speed and efficiency, theseempty drops may be removed with droplet sorting. For example, asdescribed in Example 1, a drop maker may operate close to thedripping-to-jetting transition such that, in the absence of a cell, 8 μmdrops are formed; by contrast, when a cell is present the disturbancecreated in the flow will trigger the breakup of the jet, forming drops25 μm in diameter. The device may thus produce a bi-disperse populationof empty 8 μm drops and single-cell containing 25 μm drops, which maythen be sorted by size using, e.g., a hydrodynamic sorter to recoveronly the larger, single-cell containing drops.

Passive sorters of interest include hydrodynamic sorters, which sortmicrodroplets into different channels according to size, based on thedifferent ways in which small and large drops travel through themicrofluidic channels. Also of interest are bulk sorters, a simpleexample of which is a tube containing drops of different mass in agravitational field. By centrifuging, agitating, and/or shaking thetube, lighter drops that are more buoyant will naturally migrate to thetop of the container. Drops that have magnetic properties could besorted in a similar process, except by applying a magnetic field to thecontainer, towards which drops with magnetic properties will naturallymigrate according to the magnitude of those properties. A passive sorteras used in the subject methods may also involve relatively largechannels that will sort large numbers of drops simultaneously based ontheir flow properties.

Picoinjection can also be used to change the electrical properties ofthe drops. This could be used, for example, to change the conductivityof the drops by adding ions, which could then be used to sort them, forexample, using dielectrophoresis. Alternatively, picoinjection can alsobe used to charge the drops. This could be achieved by injecting a fluidinto the drops that is charged, so that after injection, the drops wouldbe charged. This would produce a collection of drops in which some werecharged and others not, and the charged drops could then be extracted byflowing them through a region of electric field, which will deflect thembased on their charge amount. By injecting different amounts of liquidby modulating the piocoinjection, or by modulating the voltage to injectdifferent charges for affixed injection volume, the final charge on thedrops could be adjusted, to produce drops with different charge. Thesewould then be deflected by different amounts in the electric fieldregion, allowing them to be sorted into different containers.

Suitable Subjects

The subject methods may be applied to biological samples taken from avariety of different subjects. In many embodiments the subjects are“mammals” or “mammalian”, where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, andrats), and primates (e.g., humans, chimpanzees, and monkeys). In manyembodiments, the subjects are humans. The subject methods may be appliedto human subjects of both genders and at any stage of development (i.e.,neonates, infant, juvenile, adolescent, adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe present invention may be applied to a human subject, it is to beunderstood that the subject methods may also be carried-out on otheranimal subjects (that is, in “non-human subjects”) such as, but notlimited to, birds, mice, rats, dogs, cats, livestock and horses.Accordingly, it is to be understood that any subject in need ofassessment according to the present disclosure is suitable.

Moreover, suitable subjects include those who have and those who havenot been diagnosed with a condition, such as cancer. Suitable subjectsinclude those that are and are not displaying clinical presentations ofone or more cancers. In certain aspects, a subject may one that may beat risk of developing cancer, due to one or more factors such as familyhistory, chemical and/or environmental exposure, genetic mutation(s)(e.g., BRCA1 and/or BRCA2 mutation), hormones, infectious agents,radiation exposure, lifestyle (e.g., diet and/or smoking), presence ofone or more other disease conditions, and the like.

As described more fully above, a variety of different types ofbiological samples may be obtained from such subjects. In certainembodiments, whole blood is extracted from a subject. When desired,whole blood may be treated prior to practicing the subject methods, suchas by centrifugation, fractionation, purification, and the like. Thevolume of the whole blood sample that is extracted from a subject may be100 mL or less, e.g., about 100 mL or less, about 50 mL or less, about30 mL or less, about 15 mL or less, about 10 mL or less, about 5 mL orless, or about 1 mL or less.

The subject methods and devices provided herein are compatible with bothfixed and live cells. In certain embodiments, the subject methods anddevices are practiced with live cells. In other embodiments, the subjectmethods and devices are practiced with fixed cells. Fixing a cellularsample allows for the sample to be washed to extract small molecules andlipids that may interfere with downstream analysis. Further, fixing andpermeabilizing cells allows the cells to be stained with antibodies forsurface proteins as well as intracellular proteins. Combined with theRT-PCR methods described herein, such staining can be used to achievehigh levels of multiplexing because the antibodies are localized to thecell sample, while RT-PCR products are free within a microdroplet. Sucha configuration allows for dyes of the same color to be used forantibodies and for amplicons produced by RT-PCR. Any suitable method canbe used to fix cells, including but not limited to, fixing usingformaldehyde, methanol and/or acetone.

RT-PCR carried out on a fixed cell encapsulated in a microdroplet can becarried out by first diluting the microdroplet and performing the RT-PCRreaction on a sample of the diluted microdroplet. Such dilution of thecellular sample can help to limit any cellular compounds that wouldinterfere with RT-PCR. In other embodiments, the RT-PCR reagents areadded directly to the microdroplet containing the fixed cell in a “onepot” reaction without any dilution of sample. In certain embodiments,fixed cells are solubilized prior to the RT-PCR using proteases anddetergents.

Genotyping Cells

As summarized above, aspects of the invention also include methods forgenotyping components from biological samples. By “genotyping” it ismeant the detection of two or more oligonucleotides (e.g., oncogenes) ina particular cell. Aspects include methods for genotyping cells, e.g.,tumor cells, including CTCs.

In certain such aspects, the methods involve encapsulating in amicrodroplet a cell obtained from a subject's blood sample, wherein onecell is present in the microdroplet; introducing a lysing agent into themicrodroplet and incubating the microdroplet under conditions effectivefor cell lysis; introducing polymerase chain reaction (PCR) reagents anda plurality PCR primers into the microdroplet, and incubating themicrodroplet under conditions allowing for PCR amplification to producePCR amplification products, wherein the plurality of PCR primers includeone or more primers that each hybridize to one or more oncogenes;introducing a plurality of probes into the microdroplet, wherein theprobes hybridize to one or more mutations of interest and fluoresce atdifferent wavelengths; and detecting the presence or absence of specificPCR amplification products by detection of fluorescence of a probe,wherein detection of fluorescence indicates the presence of the PCRamplification products; wherein one or more of steps are performed undermicrofluidic control.

In other aspects, the methods may involve encapsulating in amicrodroplet a cell obtained from a subject's blood sample, wherein onecell is present in the microdroplet; introducing a lysing agent into themicrodroplet and incubating the microdroplet under conditions effectivefor cell lysis; introducing polymerase chain reaction (PCR) reagents anda plurality PCR primers into the microdroplet, and incubating themicrodroplet under conditions allowing for PCR amplification to producePCR amplification products, wherein the plurality of PCR primers includeone or more primers that each hybridize to one or more oncogenes, saidprimers comprising forward primers comprising a label, and reverseprimers comprising a capture sequence; introducing a fluorescent beadinto the microdroplet, wherein the bead includes a nucleotide sequencecomplementary to a capture sequence; and detecting the presence orabsence of the PCR amplification products by detection of fluorescenceof the bead and fluorescence of a primer, wherein detection offluorescence indicates the presence of the PCR amplification products;wherein one or more of steps are performed under microfluidic control.

In practicing the methods for genotyping cells, any variants to thegeneral steps described herein, such as the number of primers that maybe added, the manner in which reagents are added, suitable subjects, andthe like, may be made.

Detecting Cancer

Methods according to the present invention also involve methods fordetecting cancer. Such methods may include encapsulating in amicrodroplet oligonucleotides obtained from a biological sample from thesubject, wherein at least one oligonucleotide is present in themicrodroplet; introducing polymerase chain reaction (PCR) reagents, adetection component, and a plurality of PCR primers into themicrodroplet and incubating the microdroplet under conditions allowingfor PCR amplification to produce PCR amplification products, wherein theplurality of PCR primers include one or more primers that each hybridizeto one or more oncogenes; and detecting the presence or absence of thePCR amplification products by detection of the detection component,wherein detection of the detection component indicates the presence ofthe PCR amplification products.

Detection of one or more PCR amplification products corresponding to oneor more oncogenes may be indicative that the subject has cancer. Thespecific oncogenes that are added to the microdroplet may vary. Incertain aspects, the oncogene(s) may be specific for a particular typeof cancer, e.g., breast cancer, colon cancer, and the like.

Moreover, in practicing the subject methods the biological sample fromwhich the components are to be detected may vary, and may be based atleast in part on the particular type of cancer for which detection issought. For instance, breast tissue may be used as the biological samplein certain instances, if it is desired to determine whether the subjecthas breast cancer, and the like.

In practicing the methods for detecting cancer, any variants to thegeneral steps described herein, such as the number of primers that maybe added, the manner in which reagents are added, suitable subjects, andthe like, may be made.

Devices

As indicated above, embodiments of the invention employ microfluidicsdevices. Microfluidics devices of this invention may be characterized invarious ways. In certain embodiments, for example, microfluidics deviceshave at least one “micro” channel. Such channels may have at least onecross-sectional dimension on the order of a millimeter or smaller (e.g.,less than or equal to about 1 millimeter). Obviously for certainapplications, this dimension may be adjusted; in some embodiments the atleast one cross-sectional dimension is about 500 micrometers or less. Insome embodiments, again as applications permit, the cross-sectionaldimension is about 100 micrometers or less (or even about 10 micrometersor less—sometimes even about 1 micrometer or less). A cross-sectionaldimension is one that is generally perpendicular to the direction ofcenterline flow, although it should be understood that when encounteringflow through elbows or other features that tend to change flowdirection, the cross-sectional dimension in play need not be strictlyperpendicular to flow. It should also be understood that in someembodiments, a micro-channel may have two or more cross-sectionaldimensions such as the height and width of a rectangular cross-sectionor the major and minor axes of an elliptical cross-section. Either ofthese dimensions may be compared against sizes presented here. Note thatmicro-channels employed in this invention may have two dimensions thatare grossly disproportionate—e.g., a rectangular cross-section having aheight of about 100-200 micrometers and a width on the order or acentimeter or more. Of course, certain devices may employ channels inwhich the two or more axes are very similar or even identical in size(e.g., channels having a square or circular cross-section).

In some embodiments, microfluidic devices of this invention arefabricated using microfabrication technology. Such technology iscommonly employed to fabricate integrated circuits (ICs),microelectromechanical devices (MEMS), display devices, and the like.Among the types of microfabrication processes that can be employed toproduce small dimension patterns in microfluidic device fabrication arephotolithography (including X-ray lithography, e-beam lithography,etc.), self-aligned deposition and etching technologies, anisotropicdeposition and etching processes, self-assembling mask formation (e.g.,forming layers of hydrophobic-hydrophilic copolymers), etc.

In view of the above, it should be understood that some of theprinciples and design features described herein can be scaled to largerdevices and systems including devices and systems employing channelsreaching the millimeter or even centimeter scale channel cross-sections.Thus, when describing some devices and systems as “microfluidic,” it isintended that the description apply equally, in certain embodiments, tosome larger scale devices.

When referring to a microfluidic “device” it is generally intended torepresent a single entity in which one or more channels, reservoirs,stations, etc. share a continuous substrate, which may or may not bemonolithic. A microfluidics “system” may include one or moremicrofluidic devices and associated fluidic connections, electricalconnections, control/logic features, etc. Aspects of microfluidicdevices include the presence of one or more fluid flow paths, e.g.,channels, having dimensions as discussed herein.

In certain embodiments, microfluidic devices of this invention provide acontinuous flow of a fluid medium. Fluid flowing through a channel in amicrofluidic device exhibits many interesting properties. Typically, thedimensionless Reynolds number is extremely low, resulting in flow thatalways remains laminar. Further, in this regime, two fluids joining willnot easily mix, and diffusion alone may drive the mixing of twocompounds.

Various features and examples of microfluidic device components suitablefor use with this invention will now be described.

Substrate

Substrates used in microfluidic systems are the supports in which thenecessary elements for fluid transport are provided. The basic structuremay be monolithic, laminated, or otherwise sectioned. Commonly,substrates include one or more microchannels serving as conduits formolecular libraries and reagents (if necessary). They may also includeinput ports, output ports, and/or features to assist in flow control.

In certain embodiments, the substrate choice may be dependent on theapplication and design of the device. Substrate materials are generallychosen for their compatibility with a variety of operating conditions.Limitations in microfabrication processes for a given material are alsorelevant considerations in choosing a suitable substrate. Usefulsubstrate materials include, e.g., glass, polymers, silicon, metal, andceramics.

Polymers are standard materials for microfluidic devices because theyare amenable to both cost effective and high volume production. Polymerscan be classified into three categories according to their moldingbehavior: thermoplastic polymers, elastomeric polymers and duroplasticpolymers. Thermoplastic polymers can be molded into shapes above theglass transition temperature, and will retain these shapes after coolingbelow the glass transition temperature. Elastomeric polymers can bestretched upon application of an external force, but will go back tooriginal state once the external force is removed. Elastomers do notmelt before reaching their decomposition temperatures. Duroplasticpolymers have to be cast into their final shape because they soften alittle before the temperature reaches their decomposition temperature.

Among the polymers that may be used in microfabricated device of thisinvention are polyamide (PA), polybutylenterephthalate (PBT),polycarbonate (PC), polyethylene (PE), polymethylmethacrylate (PMMA),polyoxymethylene (POM), polypropylene (PP), polyphenylenether (PPE),polystyrene (PS) and polysulphone (PSU). The chemical and physicalproperties of polymers can limit their uses in microfluidics devices.Specifically in comparison to glass, the lower resistance againstchemicals, the aging, the mechanical stability, and the UV stability canlimit the use of polymers for certain applications.

Glass, which may also be used as the substrate material, has specificadvantages under certain operating conditions. Since glass is chemicallyinert to most liquids and gases, it is particularly appropriate forapplications employing certain solvents that have a tendency to dissolveplastics. Additionally, its transparent properties make glassparticularly useful for optical or UV detection.

Surface Treatments and Coatings

Surface modification may be useful for controlling the functionalmechanics (e.g., flow control) of a microfluidic device. For example, itmay be advantageous to keep fluidic species from adsorbing to channelwalls or for attaching antibodies to the surface for detection ofbiological components.

Polymer devices in particular tend to be hydrophobic, and thus loadingof the channels may be difficult. The hydrophobic nature of polymersurfaces also make it difficult to control electroosmotic flow (EOF).One technique for coating polymer surface is the application ofpolyelectrolyte multilayers (PEM) to channel surfaces. PEM involvesfilling the channel successively with alternating solutions of positiveand negative polyelectrolytes allowing for multilayers to formelectrostatic bonds. Although the layers typically do not bond to thechannel surfaces, they may completely cover the channels even afterlong-term storage. Another technique for applying a hydrophilic layer onpolymer surfaces involves the UV grafting of polymers to the surface ofthe channels. First grafting sites, radicals, are created at the surfaceby exposing the surface to UV irradiation while simultaneously exposingthe device to a monomer solution. The monomers react to form a polymercovalently bonded at the reaction site.

Glass channels generally have high levels of surface charge, therebycausing proteins to adsorb and possibly hindering separation processes.In some situations, it may be advantageous to apply apolydimethylsiloxane (PDMS) and/or surfactant coating to the glasschannels. Other polymers that may be employed to retard surfaceadsorption include polyacrylamide, glycol groups, polysiloxanes,glyceroglycidoxypropyl, poly(ethyleneglycol) and hydroxyethylatedpoly(ethyleneimine). Furthermore, for electroosmotic devices it isadvantageous to have a coating bearing a charge that is adjustable inmagnitude by manipulating conditions inside of the device (e.g. pH). Thedirection of the flow can also be selected based on the coating sincethe coating can either be positively or negatively charged.

Specialized coatings can also be applied to immobilize certain specieson the channel surface—this process is known by those skilled in the artas “functionalizing the surface.” For example, a polymethylmethacrylate(PMMA) surface may be coated with amines to facilitate attachment of avariety of functional groups or targets. Alternatively, PMMA surfacescan be rendered hydrophilic through an oxygen plasma treatment process.

Microfluidic Elements

Microfluidic systems can contain a number of microchannels, valves,pumps, reactors, mixers and other components. Some of these componentsand their general structures and dimensions are discussed below.

Various types of valves can be used for flow control in microfluidicdevices of this invention. These include, but are not limited to passivevalves and check valves (membrane, flap, bivalvular, leakage, etc.).Flow rate through these valves are dependent on various physicalfeatures of the valve such as surface area, size of flow channel, valvematerial, etc. Valves also have associated operational and manufacturingadvantages/disadvantages that should be taken into consideration duringdesign of a microfluidic device.

Micropumps as with other microfluidic components are subjected tomanufacturing constraints. Typical considerations in pump design includetreatment of bubbles, clogs, and durability. Micropumps currentlyavailable include, but are not limited to electric equivalent pumps,fixed-stroke microdisplacement, peristaltic micromembrane and pumps withintegrated check valves.

Macrodevices rely on turbulent forces such as shaking and stirring tomix reagents. In comparison, such turbulent forces are not practicallyattainable in microdevices, mixing in microfluidic devices is generallyaccomplished through diffusion. Since mixing through diffusion can beslow and inefficient, microstructures are often designed to enhance themixing process. These structures manipulate fluids in a way thatincreases interfacial surface area between the fluid regions, therebyspeeding up diffusion. In certain embodiments, microfluidic mixers areemployed. Such mixers may be provide upstream from (and in some casesintegrated with) a microfluidic separation device of this invention.

Micromixers may be classified into two general categories: active mixersand passive mixers. Active mixers work by exerting active control overflow regions (e.g. varying pressure gradients, electric charges, etc.).Passive mixers do not require inputted energy and use only “fluiddynamics” (e.g. pressure) to drive fluid flow at a constant rate. Oneexample of a passive mixer involves stacking two flow streams on top ofone another separated by a plate. The flow streams are contacted witheach other once the separation plate is removed. The stacking of the twoliquids increases contact area and decreases diffusion length, therebyenhancing the diffusion process. Mixing and reaction devices can beconnected to heat transfer systems if heat management is needed. As withmacro-heat exchangers, micro-heat exchanges can either have co-current,counter-current, or cross-flow flow schemes. Microfluidic devicesfrequently have channel widths and depths between about 10 μm and about10 cm. A common channel structure includes a long main separationchannel, and three shorter “offshoot” side channels terminating ineither a buffer, sample, or waste reservoir. The separation channel canbe several centimeters long, and the three side channels usually areonly a few millimeters in length. Of course, the actual length,cross-sectional area, shape, and branch design of a microfluidic devicedepends on the application as well other design considerations such asthroughput (which depends on flow resistance), velocity profile,residence time, etc.

Microfluidic devices described herein may include electric fieldgenerators to perform certain steps of the methods described herein,including, but not limited to, picoinjection, droplet coalescence,selective droplet fusion, and droplet sorting. In certain embodiments,the electric fields are generated using metal electrodes. In particularembodiments, electric fields are generated using liquid electrodes. Incertain embodiments, liquid electrodes include liquid electrode channelsfilled with a conducting liquid (e.g. salt water or buffer) and situatedat positions in the microfluidic device where an electric field isdesired. In particular embodiments, the liquid electrodes are energizedusing a power supply or high voltage amplifier. In some embodiments, theliquid electrode channel includes an inlet port so that a conductingliquid can be added to the liquid electrode channel. Such conductingliquid may be added to the liquid electrode channel, for example, byconnecting a tube filled with the liquid to the inlet port and applyingpressure. In particular embodiments, the liquid electrode channel alsoincludes an outlet port for releasing conducting liquid from thechannel. In particular embodiments, the liquid electrodes are used inpicoinjection, droplet coalescence, selective droplet fusion, and/ordroplet sorting aspects of a microfluidic device described herein.Liquid electrodes may find use, for example, where a material to beinjected via application of an electric field is not charged.

Liquid electrodes as described herein also have applicability outside ofthe specific microfluidic device applications discussed herein. Forexample, liquid electrodes may be utilized in a variety of devices inwhich metal electrodes are generally used. In addition, liquidelectrodes may be particularly well suited for use in flexible devices,such as devices that are designed to be worn on the body and/or devicesthat must flex as a result of their operation.

In certain embodiments, one or more walls of a microfluidic devicechannel immediately down-stream of a junction with one or more of aninput microchannel, pairing microchannel and/or picoinjectionmicrochannel includes one or more ridges. Such ridges in the walls ofthe microchannel are configured to trap a layer of a suitable phase,e.g., a suitable hydrophobic phase (e.g., oil) and thereby prevent animmiscible phase, e.g., an aqueous phase, from touching the walls of themicrochannel, which can cause wetting of the channel walls. Such wettingmay be undesirable as it may lead to unpredictable drop formation and/orallow fluids to transfer between drops, leading to contamination. Incertain embodiments, the ridges allow for the formation of drops athigher flow rate ratios R (Q_(aq)/Q_(sum)).

In certain embodiments, the width of one or more of the microchannels ofthe microfluidic device (e.g., input microchannel, pairing microchannel,picoinjection microchannel, and/or a flow channel upstream or downstreamof one or more of these channels) is 100 microns or less, e.g., 90microns or less, 80 microns or less, 70 microns or less, 60 microns orless, 50 microns or less, e.g., 45 microns or less, 40 microns or less,39 microns or less, 38 microns or less, 37 microns or less, 36 micronsor less, 35 microns or less, 34 microns or less, 33 microns or less, 32microns or less, 31 microns or less, 30 microns or less, 29 microns orless, 28 microns or less, 27 microns or less, 26 microns or less, 25microns or less, 20 microns or less, 15 microns or less, or 10 micronsor less. In some embodiments, the width of one or more of the abovemicrochannels is from about 10 microns to about 15 microns, from about15 microns to about 20 microns, from about 20 microns to about 25microns, from about 25 microns to about 30 microns, from about 30microns to about 35 microns, from about 35 microns to about 40 microns,from about 40 microns to about 45 microns, or from about 45 microns toabout 50 microns, from about 50 microns to about 60 microns, from about60 microns to about 70 microns, from about 70 microns to about 80microns, from about 80 microns to about 90 microns, or from about 90microns to about 100 microns.

In certain embodiments, the base of each of the one or more ridges isfrom about 10 microns to about 20 microns in length, e.g., from about 11to about 19 microns in length, from about 12 to about 18 microns inlength, from about 13 to about 17 microns in length, from about 14 toabout 16 microns in length, or about 15 microns in length.

In certain embodiments, the peak of each of the one or more ridges has awidth of about 1 to about 10 microns, e.g., from about 1 to about 9microns, from about 2 to about 8 microns, from about 3 to about 7microns, from about 4 to about 6 microns, or about 5 microns. In certainembodiments, the peak of each of the one or more ridges has a width offrom about 1 micron to about 2 microns, from about 2 microns to about 3microns, from about 3 microns to about 4 microns, from about 4 micronsto about 5 microns, from about 5 microns to about 6 microns, from about6 microns to about 7 microns, from about 7 microns to about 8 microns,from about 8 microns to about 9 microns, or from about 9 microns toabout 10 microns.

In certain embodiments, the height of each of the one or more ridges isfrom about 5 microns to about 15 microns, e.g., about 6 microns to about14 microns, about 7 microns to about 13 microns, about 8 microns toabout 12 microns, about 9 microns to about 11 microns, or about 10microns.

In certain embodiments, the ratio of the base of each of the one or moreridges to the height of each of the one or more ridges is from about1.0:0.75 to about 0.75:1.0. In certain embodiments, the ratio of thebase of each of the one or more ridges to the width of the peak of eachof the one or more ridges is about 1.0:0.5 to about 1.0:0.1, e.g, fromabout 1.0:0.2, from about 1.0:0.3, or from about 1.0:0.4.

In certain embodiments, the ratio of the base of each of the one or moreridges to the height of each of the one or more ridges to the width ofthe peak of the one or more ridges is about 1:0.75:0.5.

In certain embodiments, a channel as described herein is provided with aplurality of ridges which extend for a distance along the channel wall.This distance may be, for example, from about 50 microns to about 500microns, e.g., from about 50 microns to about 450 microns, from about100 microns to about 400 microns, from about 150 microns to about 350microns, from about 200 microns to about 300 microns, or about 250microns. In certain embodiments, a plurality of ridges may be providedwhich extend for a distance along the channel wall, wherein the ratiobetween the distance along the channel wall and the width of the channelis from about 10:1 to about 1:2, e.g., about 10:1, about 9:1, about 8:1,about 7:1, about 6:1 about 5:1, about 4:1, about 3:1, about 2:1, about1:1, or about 1:2.

It should be noted that one or more of the various dimensions discussedabove may be scaled up or down as appropriate for a particularapplication, for example each of the above dimensions may be scaled upor down by a factor of 2, 5, 10 or more as appropriate.

In some embodiments, one or more channel junctions, e.g., one or moredroplet forming junctions, such as a picoinjector junction, include a“step-down” structure. This is depicted, for example, in FIG. 26,wherein the portion of the flow channel at the picoinjector junction anddownstream of the picoinjector junction is wider than the portion of theflow channel upstream of the picoinjector junction. This step-downstructure facilitates the pinching-off of droplets and thus facilitatesdroplet formation. The step size may be chosen based on the desired sizeof the droplet to be formed, with larger steps creating larger droplets.Such structures may also help to avoid dripping of material from thepicoinjector following injection from the picoinjector into a droplet.In some embodiments, the width of the flow channel at the picoinjectorjunction and downstream of the picoinjector junction is from about 5% toabout 50% wider than the width of the flow channel immediately upstreamof the picoinjector junction, e.g., about 5 to about 10% wider, about 10to about 20% wider, about 20 to about 30% wider, about 30 to about 40%wider or about 40 to about 50% wider.

Methods of Fabrication

Microfabrication processes differ depending on the type of materialsused in the substrate and the desired production volume. For smallvolume production or prototypes, fabrication techniques include LIGA,powder blasting, laser ablation, mechanical machining, electricaldischarge machining, photoforming, etc. Technologies for mass productionof microfluidic devices may use either lithographic or master-basedreplication processes. Lithographic processes for fabricating substratesfrom silicon/glass include both wet and dry etching techniques commonlyused in fabrication of semiconductor devices. Injection molding and hotembossing typically are used for mass production of plastic substrates.

Glass, Silicon and Other “Hard” Materials (Lithography, Etching,Deposition)

The combination of lithography, etching and deposition techniques may beused to make microcanals and microcavities out of glass, silicon andother “hard” materials. Technologies based on the above techniques arecommonly applied in for fabrication of devices in the scale of 0.1-500micrometers.

Microfabrication techniques based on current semiconductor fabricationprocesses are generally carried out in a clean room. The quality of theclean room is classified by the number of particles<4 μm in size in acubic inch. Typical clean room classes for MEMS microfabrication are1000 to 10000.

In certain embodiments, photolithography may be used inmicrofabrication. In photolithography, a photoresist that has beendeposited on a substrate is exposed to a light source through an opticalmask. Conventional photoresist methods allow structural heights of up to10-40 μm. If higher structures are needed, thicker photoresists such asSU-8, or polyimide, which results in heights of up to 1 mm, can be used.

After transferring the pattern on the mask to the photoresist-coveredsubstrate, the substrate is then etched using either a wet or dryprocess. In wet etching, the substrate—area not protected by themask—subjected to chemical attack in the liquid phase. The liquidreagent used in the etching process depends on whether the etching isisotropic or anisotropic. Isotropic etching generally uses an acid toform three-dimensional structures such as spherical cavities in glass orsilicon. Anisotropic etching forms flat surfaces such as wells andcanals using a highly basic solvent. Wet anisotropic etching on siliconcreates an oblique channel profile.

Dry etching involves attacking the substrate by ions in either a gaseousor plasma phase. Dry etching techniques can be used to createrectangular channel cross-sections and arbitrary channel pathways.Various types of dry etching that may be employed including physical,chemical, physico-chemical (e.g., RIE), and physico-chemical withinhibitor. Physical etching uses ions accelerated through an electricfield to bombard the substrate's surface to “etch” the structures.Chemical etching may employ an electric field to migrate chemicalspecies to the substrate's surface. The chemical species then reactswith the substrate's surface to produce voids and a volatile species.

In certain embodiments, deposition is used in microfabrication.Deposition techniques can be used to create layers of metals,insulators, semiconductors, polymers, proteins and other organicsubstances. Most deposition techniques fall into one of two maincategories: physical vapor deposition (PVD) and chemical vapordeposition (CVD). In one approach to PVD, a substrate target iscontacted with a holding gas (which may be produced by evaporation forexample). Certain species in the gas adsorb to the target's surface,forming a layer constituting the deposit. In another approach commonlyused in the microelectronics fabrication industry, a target containingthe material to be deposited is sputtered with using an argon ion beamor other appropriately energetic source. The sputtered material thendeposits on the surface of the microfluidic device. In CVD, species incontact with the target react with the surface, forming components thatare chemically bonded to the object. Other deposition techniquesinclude: spin coating, plasma spraying, plasma polymerization, dipcoating, casting and Langmuir-Blodgett film deposition. In plasmaspraying, a fine powder containing particles of up to 100 μm in diameteris suspended in a carrier gas. The mixture containing the particles isaccelerated through a plasma jet and heated. Molten particles splatteronto a substrate and freeze to form a dense coating. Plasmapolymerization produces polymer films (e.g. PMMA) from plasma containingorganic vapors.

Once the microchannels, microcavities and other features have beenetched into the glass or silicon substrate, the etched features areusually sealed to ensure that the microfluidic device is “watertight.”When sealing, adhesion can be applied on all surfaces brought intocontact with one another. The sealing process may involve fusiontechniques such as those developed for bonding between glass-silicon,glass-glass, or silicon-silicon.

Anodic bonding can be used for bonding glass to silicon. A voltage isapplied between the glass and silicon and the temperature of the systemis elevated to induce the sealing of the surfaces. The electric fieldand elevated temperature induces the migration of sodium ions in theglass to the glass-silicon interface. The sodium ions in theglass-silicon interface are highly reactive with the silicon surfaceforming a solid chemical bond between the surfaces. The type of glassused should ideally have a thermal expansion coefficient near that ofsilicon (e.g. Pyrex Corning 7740).

Fusion bonding can be used for glass-glass or silicon-silicon sealing.The substrates are first forced and aligned together by applying a highcontact force. Once in contact, atomic attraction forces (primarily vander Waals forces) hold the substrates together so they can be placedinto a furnace and annealed at high temperatures. Depending on thematerial, temperatures used ranges between about 600 and 1100° C.

Polymers/Plastics

A number of techniques may be employed for micromachining plasticsubstrates in accordance with embodiments of this invention. Among theseare laser ablation, stereolithography, oxygen plasma etching, particlejet ablation, and microelectro-erosion. Some of these techniques can beused to shape other materials (glass, silicon, ceramics, etc.) as well.

To produce multiple copies of a microfluidic device, replicationtechniques are employed. Such techniques involve first fabricating amaster or mold insert containing the pattern to be replicated. Themaster is then used to mass-produce polymer substrates through polymerreplication processes.

In the replication process, the master pattern contained in a mold isreplicated onto the polymer structure. In certain embodiments, a polymerand curing agent mix is poured onto a mold under high temperatures.After cooling the mix, the polymer contains the pattern of the mold, andis then removed from the mold. Alternatively, the plastic can beinjected into a structure containing a mold insert. In microinjection,plastic heated to a liquid state is injected into a mold. Afterseparation and cooling, the plastic retains the mold's shape.

PDMS (polydimethylsiloxane), a silicon-based organic polymer, may beemployed in the molding process to form microfluidic structures. Becauseof its elastic character, PDMS is well suited for microchannels betweenabout 5 and 500 μm. Specific properties of PDMS make it particularlysuitable for microfluidic purposes:

-   -   1) It is optically clear which allows for visualization of the        flows;    -   2) PDMS when mixed with a proper amount of reticulating agent        has elastomeric qualities that facilitates keeping microfluidic        connections “watertight;”    -   3) Valves and pumps using membranes can be made with PDMS        because of its elasticity;    -   4) Untreated PDMS is hydrophobic, and becomes temporarily        hydrophilic after oxidation of surface by oxygen plasma or after        immersion in strong base; oxidized PDMS adheres by itself to        glass, silicon, or polyethylene, as long as those surfaces were        themselves exposed to an oxygen plasma.    -   5) PDMS is permeable to gas. Filling of the channel with liquids        is facilitated even when there are air bubbles in the canal        because the air bubbles are forced out of the material. But it's        also permeable to non polar-organic solvents.

Microinjection can be used to form plastic substrates employed in a widerange of microfluidic designs. In this process, a liquid plasticmaterial is first injected into a mold under vacuum and pressure, at atemperature greater than the glass transition temperature of theplastic. The plastic is then cooled below the glass transitiontemperature. After removing the mold, the resulting plastic structure isthe negative of the mold's pattern.

Yet another replicating technique is hot embossing, in which a polymersubstrate and a master are heated above the polymer's glass transitiontemperature, Tg (which for PMMA or PC is around 100-180° C.). Theembossing master is then pressed against the substrate with a presetcompression force. The system is then cooled below Tg and the mold andsubstrate are then separated.

Typically, the polymer is subjected to the highest physical forces uponseparation from the mold tool, particularly when the microstructurecontains high aspect ratios and vertical walls. To avoid damage to thepolymer microstructure, material properties of the substrate and themold tool may be taken into consideration. These properties include:sidewall roughness, sidewall angles, chemical interface betweenembossing master and substrate and temperature coefficients. Highsidewall roughness of the embossing tool can damage the polymermicrostructure since roughness contributes to frictional forces betweenthe tool and the structure during the separation process. Themicrostructure may be destroyed if frictional forces are larger than thelocal tensile strength of the polymer. Friction between the tool and thesubstrate may be important in microstructures with vertical walls. Thechemical interface between the master and substrate could also be ofconcern. Because the embossing process subjects the system to elevatedtemperatures, chemical bonds could form in the master-substrateinterface. These interfacial bonds could interfere with the separationprocess. Differences in the thermal expansion coefficients of the tooland the substrate could create addition frictional forces.

Various techniques can be employed to form molds, embossing masters, andother masters containing patterns used to replicate plastic structuresthrough the replication processes mentioned above. Examples of suchtechniques include LIGA (described below), ablation techniques, andvarious other mechanical machining techniques. Similar techniques canalso be used for creating masks, prototypes and microfluidic structuresin small volumes. Materials used for the mold tool include metals, metalalloys, silicon and other hard materials.

Laser ablation may be employed to form microstructures either directlyon the substrate or through the use of a mask. This technique uses aprecision-guided laser, typically with wavelength between infrared andultraviolet. Laser ablation may be performed on glass and metalsubstrates, as well as on polymer substrates. Laser ablation can beperformed either through moving the substrate surface relative to afixed laser beam, or moving the beam relative to a fixed substrate.Various micro-wells, canals, and high aspect structures can be made withlaser ablation.

Certain materials such as stainless steel make very durable mold insertsand can be micromachined to form structures down to the 10-μm range.Various other micromachining techniques for microfabrication existincluding μ-Electro Discharge Machining (μ-EDM), μ-milling, focused ionbeam milling. μ-EDM allows the fabrication of 3-dimensional structuresin conducting materials. In μ-EDM, material is removed by high-frequencyelectric discharge generated between an electrode (cathode tool) and aworkpiece (anode). Both the workpiece and the tool are submerged in adielectric fluid. This technique produces a comparatively roughersurface but offers flexibility in terms of materials and geometries.

Electroplating may be employed for making a replication mold tool/masterout of, e.g., a nickel alloy. The process starts with a photolithographystep where a photoresist is used to defined structures forelectroplating. Areas to be electroplated are free of resist. Forstructures with high aspect ratios and low roughness requirements, LIGAcan be used to produce electroplating forms. LIGA is a German acronymfor Lithographic (Lithography), Galvanoformung (electroplating),Abformung (molding). In one approach to LIGA, thick PMMA layers areexposed to x-rays from a synchrotron source. Surfaces created by LIGAhave low roughness (around 10 nm RMS) and the resulting nickel tool hasgood surface chemistry for most polymers.

As with glass and silicon devices, polymeric microfluidic devices mustbe closed up before they can become functional. Common problems in thebonding process for microfluidic devices include the blocking ofchannels and changes in the physical parameters of the channels.Lamination is one method used to seal plastic microfluidic devices. Inone lamination process, a PET foil (about 30 μm) coated with a meltingadhesive layer (typically 5-10 μm) is rolled with a heated roller, ontothe microstructure. Through this process, the lid foil is sealed ontothe channel plate. Several research groups have reported a bonding bypolymerization at interfaces, whereby the structures are heated andforce is applied on opposite sides to close the channel. But excessiveforce applied may damage the microstructures. Both reversible andirreversible bonding techniques exist for plastic-plastic andplastic-glass interfaces. One method of reversible sealing involvesfirst thoroughly rinsing a PDMS substrate and a glass plate (or a secondpiece of PDMS) with methanol and bringing the surfaces into contact withone another prior to drying. The microstructure is then dried in an ovenat 65° C. for 10 min. No clean room is required for this process.Irreversible sealing is accomplished by first thoroughly rinsing thepieces with methanol and then drying them separately with a nitrogenstream. The two pieces are then placed in an air plasma cleaner andoxidized at high power for about 45 seconds. The substrates are thenbrought into contact with each other and an irreversible seal formsspontaneously.

Other available techniques include laser and ultrasonic welding. Inlaser welding, polymers are joined together through laser-generatedheat. This method has been used in the fabrication of micropumps.Ultrasonic welding is another bonding technique that may be employed insome applications.

The nucleic acid amplification technique described here is a polymerasechain reaction (PCR). However, in certain embodiments, non-PCRamplification techniques may be employed such as various isothermalnucleic acid amplification techniques; e.g., real-time stranddisplacement amplification (SDA), rolling-circle amplification (RCA) andmultiple-displacement amplification (MDA).

Regarding PCR amplification modules, it will be necessary to provide tosuch modules at least the building blocks for amplifying nucleic acids(e.g., ample concentrations of four nucleotides), primers, polymerase(e.g., Taq), and appropriate temperature control programs). Thepolymerase and nucleotide building blocks may be provided in a buffersolution provided via an external port to the amplification module orfrom an upstream source. In certain embodiments, the buffer streamprovided to the sorting module contains some of all the raw materialsfor nucleic acid amplification. For PCR in particular, precisetemperature control of the reacting mixture is extremely important inorder to achieve high reaction efficiency. One method of on-chip thermalcontrol is Joule heating in which electrodes are used to heat the fluidinside the module at defined locations. The fluid conductivity may beused as a temperature feedback for power control.

In certain aspects, the drops containing the PCR mix may be flowedthrough a channel that incubates the droplets under conditions effectivefor PCR. Flowing the microdroplets through a channel may involve achannel that snakes over various temperature zones maintained attemperatures effective for PCR. Such channels may, for example, cycleover two or more temperature zones, wherein at least one zone ismaintained at about 65° C. and at least one zone is maintained at about95° C. As the drops move through such zones, their temperature cycles,as needed for PCR. The precise number of zones, and the respectivetemperature of each zone, may be readily determined by those of skill inthe art to achieve the desired PCR amplification.

In other embodiments, incubating the microdroplets may involve the useof a Megadroplet Array. In such a device, an array consists of channelsin which the channel ceilings are indented with millions of circulartraps that are about 25 μm in diameter. Drops are distributed into thetrapping channels using distribution plates—large channels connectingthe inlets of the trapping channels (FIG. 12, Panel A). Due to the largesize of the distribution channels compared to the trapping channels—thedistribution channels are about 100×500 μm in height and width, comparedto only about 15×100 μm for the droplet trapping channels—thehydrodynamic resistance of the distribution channels is ˜1500 timeslower than that of the trapping channels; this ensures that thedistribution channel fills with drops before the trapping channels beginto fill, allowing even distribution of the drops into the trappingchannels. When the drops flow into the trapping channels, they areslightly pancaked in shape because the vertical height of the channel is15 μm, or 10 μm shorter than the drops, as illustrated in FIG. 12, PanelB. When a drop nears a trap, its interface adopts a larger, moreenergetically favorable radius of curvature. To minimize its surfaceenergy, the drop entirely fills the trap, allowing it to adopt thelowest, most energetically favorable, average radius of curvature. Aftera trap is occupied by a drop, no other drops are able to enter becausethe trap is large enough to fit only one drop; additional drops arediverted downstream, to occupy the first vacant trap they encounter.Because the array is filled using a close-packed emulsion, every trapwill be occupied by a drop, since this is the most energeticallyfavorable state under low flow conditions. After the droplet array isfilled, oil is injected to remove excess drops and the array is thermalcycled and imaged.

A variety of different ways can be used to fill the traps of the device.For instance, buoyancy effects and centrifugation can also be used tofill and empty the traps by flipping the device with respect to theearth's gravitational field, since the droplet density is 63% that ofthe fluorocarbon carrier oil. That is, if the drops were heavier thanthe oil phase, then the wells could be imprinted into the “floor” of thedevice so that when the emulsion was flowed over it, the drops wouldsink into the wells. The flow rate of the emulsion could be adjusted tooptimize this and the drop size would be made to be approximately thesame size as the well so that the well could only fit a single drop at atime. In other aspects, the drops could also, or instead, be stored in alarge chamber with no wells.

The device may achieve thermal cycling using a heater consisting of aPeltier plate, heat sink, and control computer (FIG. 12, Panel A; FIG.13). The Peltier plate allows heating and/or cooling the chip above orbelow room temperature by controlling the applied current. To ensurecontrolled and reproducible temperature, a computer monitors thetemperature of the array using integrated temperature probes, andadjusts the applied current to heat and cool as needed. A metallic(e.g., copper) plate allows uniform application of heat and dissipationof excess heat during cooling cycles, enabling cooling from 95° C. to60° C. in under 1 min execution. In order to image microdroplets,certain embodiments may incorporate a scanner bed. In certain aspects,the scanner bed is a Canoscan 9000F scanner bed.

In order to effectively amplify nucleic acids from target components,the microfluidics system may include a cell lysing or viral proteincoat-disrupting module to free nucleic acids prior to providing thesample to an amplification module. Cell lysing modules may rely onchemical, thermal, and/or mechanical means to effect cell lysis. Becausethe cell membrane consists of a lipid double-layer, lysis bufferscontaining surfactants can solubilize the lipid membranes. Typically,the lysis buffer will be introduced directly to a lysis chamber via anexternal port so that the cells are not prematurely lysed during sortingor other upstream process. In cases where organelle integrity isnecessary, chemical lysis methods may be inappropriate. Mechanicalbreakdown of the cell membrane by shear and wear is appropriate incertain applications. Lysis modules relying mechanical techniques mayemploy various geometric features to effect piercing, shearing,abrading, etc. of cells entering the module. Other types of mechanicalbreakage such as acoustic techniques may also yield appropriate lysate.Further, thermal energy can also be used to lyse cells such as bacteria,yeasts, and spores. Heating disrupts the cell membrane and theintracellular materials are released. In order to enable subcellularfractionation in microfluidic systems a lysis module may also employ anelectrokinetic technique or electroporation. Electroporation createstransient or permanent holes in the cell membranes by application of anexternal electric field that induces changes in the plasma membrane anddisrupts the transmembrane potential. In microfluidic electroporationdevices, the membrane may be permanently disrupted, and holes on thecell membranes sustained to release desired intracellular materialsreleased.

Single Cell RT-PCR Microfluidic Device

In another aspect, provided herein is a single cell RT-PCR microfluidicdevice, described in greater detail below with reference to FIG. 32. Incertain embodiments, the single cell RT-PCR microfluidic device includesan input microchannel, which may be coupled to a flow focus drop maker,for introducing microdroplets into the microfluidic device, wherein theflow focus drop maker spaces the microdroplets in the inputmicrochannel, e.g., by a volume of a suitable hydrophobic phase, e.g.,oil, wherein each microdroplet may include a cell lysate sample. Anexemplary embodiment is shown in FIG. 32 (Panel A).

The microfluidic device may further include a pairing microchannel influidic communication with the input microchannel and a dilution bufferdrop maker in fluidic communication with the pairing microchannel. Insuch embodiments, a microdroplet from the input microchannel flows intothe pairing microchannel where the dilution buffer drop maker produces adrop of dilution buffer that is larger than and paired with eachmicrodroplet. In certain embodiments, the dilution buffer drop maker isa T-junction drop maker. An exemplary embodiment is shown in FIG. 32(Panel B).

The microfluidic device may also include a merging microchannel influidic communication with the pairing microchannel, the mergingmicrochannel including an electric field generator positioned inproximity thereto. In such embodiments, the paired microdroplet and dropof dilution buffer enter the merging microchannel from the pairingmicrochannel and are merged upon passing through an electric fieldproduced by the electric field generator to produce a dilutedmicrodroplet. Any suitable electric field generator can be used toproduce the diluted microdroplet. In certain embodiments, the electricfield is created by metal electrodes. In other embodiments, the electricfield is created by liquid electrodes as discussed herein. An exemplaryembodiment is shown in FIG. 32 (Panel C).

The microfluidic device may also include a series of mixingmicrochannels in fluidic communication with the merging microchannel.Such mixing microchannels allow for the mixing of the contents of thediluted microdroplet.

The microfluidic device may also include a drop sampler in fluidiccommunication with the mixing microchannels. Such a drop sampler iscapable of taking a sample of the diluted microdroplet, e.g., to be usedin a subsequent RT-PCR reaction carried out in the microfluidic device.An exemplary embodiment is shown in FIG. 32 (Panel D).

The microfluidic may also include a picoinjection microchannelcomprising a picoinjector, wherein the picoinjection microchannel may bea pressurized microchannel capable of receiving the sample of thediluted microdroplet produced by the drop sampler and allowing thepicoinjector to picoinject RT-PCR reagents into the sample. In certainembodiments the picoinjection is assisted by an electric field appliedto the picoinjection microchannel. Any electric field generator can beused to create an electric field for picoinjection. In certainembodiments, the electric field is created by metal electrodes. In otherembodiments, the electric field is created by liquid electrodes asdiscussed herein. An exemplary embodiment is shown in FIG. 32 (Panel E).

Samples of the diluted microdroplet that have been picoinjected withRT-PCR reagents can then be subjected to conditions for RT-PCR using anyof the approaches described herein. The single cell RT-PCR microfluidicdevice advantageously allows for the dilution of the cell lysate sampleprior to addition of RT-PCR agents. Such dilution helps in preventinhibition of RT-PCR that may be caused by components of the celllysate. In certain embodiments, the microfluidic device also includes anencapsulating chamber in fluidic communication with the inputmicrochannel, for encapsulating a cell and lysis reagent into amicrodroplet. In such embodiments, the input microchannel is capable ofreceiving the microdroplet from the encapsulating chamber.

Certain non-limiting aspects of the disclosure are provided below:

-   -   1. A method for the detection of cells, the method including:        -   encapsulating in a microdroplet a cell obtained from a            biological sample from a subject, wherein at least one cell            is present in the microdroplet;        -   incubating the microdroplet under conditions effective for            cell lysis;        -   introducing polymerase chain reaction (PCR) reagents, a            detection component, and a plurality of PCR primers into the            microdroplet and incubating the microdroplet under            conditions allowing for PCR amplification to produce PCR            amplification products, wherein the plurality of PCR primers            include one or more primers that each hybridize to one or            more oligonucleotides; and        -   detecting the presence or absence of the PCR amplification            products by detection of the detection component, wherein            detection of the detection component indicates the presence            of PCR amplification products;        -   wherein one or more steps are performed under microfluidic            control.    -   2. The method according to 1, wherein incubating the        microdroplet under conditions effective for cell lysis includes        introducing a lysing agent into the microdroplet.    -   3. The method according to 1 or 2, wherein the one or more        oligonucleotides are oncogenes.    -   4. The method according to any of 1-3, wherein the biological        sample is blood and the method includes determining the number        of circulating tumor cells (CTCs) present in the sample of the        subject's blood based at least in part on the number of        microdroplets in which PCR amplification products were detected.    -   5. The method according to any of 1-4, wherein all steps are        performed under microfluidic control.    -   6. The method according to 5, wherein all steps are performed on        the same microfluidic device.    -   7. The method according to any of the above, wherein the        plurality of PCR primers includes 10 or more primers.    -   8. The method according to any of the above, wherein the        plurality of PCR primers includes 20 to 100 primers.    -   9. The method according to any of the above, wherein the        plurality of PCR primers includes primers for 10 or more        oncogenes.    -   10. The method according to any of the above, wherein incubating        the microdroplet under conditions allowing for PCR amplification        is performed on the same microfluidic device used to encapsulate        the cells and lyse the cells.    -   11. The method according to any of the above, wherein the PCR        reagents and PCR primers are added at the same time as the        lysing agent.    -   12. The method according to any of the above, wherein the PCR        reagents are added in two steps or more.    -   13. The method according to any of the above, further including        introducing a probe into the microdroplet.    -   14. The method according to 13, wherein the probe is introduced        prior to incubating the microdroplet under conditions allowing        for PCR amplification.    -   15. The method according to 13 or 14, wherein the probe is a        TaqMan® probe.    -   16. The method according to any of the above, wherein a reagent        is added to the microdroplet by merging the microdroplet with a        second microdroplet including the reagent.    -   17. The method according to any of the above, wherein a reagent        is added to the microdroplet using either droplet coalescence or        picoinjection.    -   18. The method according to any of the above, wherein a reagent        is added to the microdroplet by a method including:        -   a) emulsifying the reagent into a stream of droplets,            wherein the droplets are smaller than the size of the            microdroplet;        -   b) flowing the droplets together with the microdroplet; and        -   c) merging a droplet with the microdroplet.    -   19. The method according to 18, wherein the diameter of the        droplets is 25% or less than that of the diameter of the        microdroplet, and a plurality of droplets are merged with the        microdroplet.    -   20. The method according to 18 or 19, wherein the merging        includes applying an electric field.    -   21. The method according to any of the above, wherein a reagent        is added to the microdroplet by a method including:        -   a) jetting the reagent into a fluid jet;        -   b) flowing the fluid jet alongside the microdroplet; and        -   c) merging a droplet with the microdroplet.    -   22. The method according to 21, wherein merging includes        applying an electric field.    -   23. The method according to 21 or 22, wherein jetting the        reagent includes adding a viscosity-increasing agent or        surfactant.    -   24. The method according to any of the above, wherein a reagent        is added to the microdroplet by a method including using a fluid        injected into the microdroplet as an electrode.    -   25. The method according to any of the above, wherein the        detection component is detected based on a change in        fluorescence.    -   26. The method according to 25, where in the change in        fluorescence is due to fluorescence resonance energy transfer        (FRET).    -   27. The method according to 25, where in the change in        fluorescence is due to fluorescence polarization.    -   28. The method according to 25 or 27, wherein the detection        component is an intercalating stain.    -   29. The method according to any of the above, wherein detecting        the presence or absence of the PCR amplification products        includes repeatedly imaging the microdroplet.    -   30. The method according to 29, wherein the microdroplet is        repeatedly imaged while the microdroplet is subjected to        conditions allowing for PCR amplification to produce the PCR        amplification products.    -   31. The method according to any of the above, wherein incubating        the microdroplet under conditions allowing for PCR amplification        and detecting the presence or absence of the PCR amplification        products are performed on a Megadroplet Array.    -   32. The method according to any of the above, including sorting        a microdroplet.    -   33. The method according to 32, wherein the sorting includes        using membrane valves, bifurcating channels, surface acoustic        waves, or dielectrophoresis.    -   34. The method according to 32 or 33, wherein the microdroplet        is sorted based on a property including size, viscosity, mass,        buoyancy, surface tension, electrical conductivity, charge, or        magnetism.    -   35. The method according to any of 32-34, including sorting        based at least in part based upon the detection of the presence        or absence of PCR amplification products.    -   36. The method according to any of 32-35, wherein the        microdroplet is sorted prior to the introduction of a PCR        reagent.    -   37. The method according to any of 32-36, wherein the        microdroplet is sorted prior to the introduction of a lysing        agent.    -   38. The method according to any of the above, further including:        -   injecting a diluent into the microdroplet; and        -   flowing the microdroplet through a microfluidic channel on            which an electric field is being applied, under conditions            in which the microdroplet is split.    -   39. The method according to any of the above, wherein the        subject is mammalian.    -   40. The method according to any of the above, wherein the        subject is human.    -   41. The method according to any of the above, wherein the        subject has been diagnosed with cancer.    -   42. The method according to any of the above, wherein the        biological sample is a blood sample.    -   43. The method of 42, wherein the blood sample is whole blood.    -   44. The method of 42 or 43, including fractionating the blood        sample.    -   45. The method of any one of 42-44, including drawing 30 mL or        less of the subject's blood.    -   46. The method of 45, wherein the blood sample is 15 mL or less.    -   47. The method of any one of the above, including fixing and/or        permeabilizing the cell.    -   48. The method of any one of the above, including introducing a        plurality of different detection components, and detecting the        presence or absence of the PCR amplification products by        detection of the plurality of detection components, wherein        detection of the detection components indicates the presence of        PCR amplification products.    -   49. The method of any one of the above, including contacting the        cell or a component thereof with a detectably labeled antibody.    -   50. A method for the detection of tumor cells, the method        including:        -   encapsulating a plurality of cells in a plurality of            microdroplets under conditions in which a majority of            microdroplets include zero or one cell, wherein the            plurality of cells are obtained from a subject's blood            sample suspected of containing circulating tumor cells            (CTCs);        -   enriching the plurality of microdroplets for microdroplets            containing one cell;        -   introducing a lysing agent into the plurality of            microdroplets and incubating under conditions effective for            cell lysis;        -   introducing polymerase chain reaction (PCR) reagents, a            detection component, and a plurality of PCR primers into the            plurality of microdroplets and incubating the plurality of            microdroplets under conditions allowing for PCR            amplification to produce PCR amplification products, wherein            the plurality of PCR primers include one or more primers            that each hybridize to one or more oncogenes;        -   detecting the presence or absence of the PCR amplification            products by detection of the detection component, wherein            detection of the detection component indicates the presence            of the PCR amplification products; and        -   determining the number of CTCs present in a sample of the            subject's blood based at least in part on the number of            microdroplets in which the PCR amplification products were            detected;        -   wherein one or more steps are performed under microfluidic            control.    -   51. The method according to 50, wherein all steps are all        performed under microfluidic control.    -   52. The method according to 50 or 51, wherein all steps are        performed on the same microfluidic device.    -   53. The method according to any of 50-52, wherein the plurality        of PCR primers includes 10 or more primers.    -   54. The method according to any of 50-53, wherein the plurality        of PCR primers includes primers for 10 or more oncogenes.    -   55. The method according to any of 50-54, wherein the plurality        of PCR primers includes a plurality of probes.    -   56. The method according to 55, wherein the probes include        TaqMan® probes.    -   57. The method according to any of 50-56, wherein the PCR        reagents are added in two steps or more.    -   58. The method according to any of 50-57, further including        introducing a probe into the microdroplet.    -   59. The method according to any of 50-58, wherein a reagent is        added to the plurality of microdroplets by merging a        microdroplet with a second microdroplet including the reagent.    -   60. The method according to any of 50-59, wherein a reagent is        added to the plurality of microdroplets using either droplet        coalescence or picoinjection.    -   61. The method according to any of 50-60, wherein a reagent is        added to the plurality of microdroplets by a method including:        -   a) emulsifying the reagent into a stream of droplets,            wherein the droplets are smaller than the size of a            microdroplet;        -   b) flowing the droplets together with the microdroplet; and        -   c) merging a droplet with the microdroplet.    -   62. The method according to 58, wherein the merging includes        applying an electric field.    -   63. The method according to any of 50-62, wherein a reagent is        added to the plurality of microdroplets by a method including:        -   a) jetting the reagent into a fluid jet;        -   b) flowing the fluid jet alongside a microdroplet; and        -   c) merging a droplet with the microdroplet.    -   64. The method according to any of 50-63, wherein a reagent is        added to the microdroplet by a method including using a fluid        injected into the microdroplet as an electrode.    -   65. The method according to any of 50-64, including sorting a        microdroplet.    -   66. The method according to 65, wherein the plurality of        microdroplets is sorted based on a property including size,        viscosity, mass, buoyancy, surface tension, electrical        conductivity, charge, or magnetism.    -   67. The method according to any of 65-66, wherein the plurality        of microdroplets is sorted prior to the introduction of a PCR        reagent.    -   68. The method according to any of 50-67, wherein detecting the        presence or absence of the PCR amplification products includes        repeatedly imaging the plurality of microdroplets.    -   69. The method according to 68, wherein the plurality of        microdroplets is repeatedly imaged while the plurality of        microdroplets is subjected to conditions allowing for PCR        amplification to produce the PCR amplification products.    -   70. The method according to any of 50-69, wherein incubating the        plurality of microdroplets under conditions allowing for PCR        amplification and detecting the presence or absence of the PCR        amplification products are performed on a Megadroplet Array.    -   71. The method according to any of 50-70, wherein the subject is        mammalian.    -   72. The method according to any of 50-71, wherein the subject is        human.    -   73. The method according to any of 50-72, wherein the subject        has been diagnosed with cancer.    -   74. A method for genotyping of cells, the method including:        -   encapsulating in a microdroplet a cell obtained from a            biological sample from a subject, wherein one cell is            present in the microdroplet;        -   introducing a lysing agent into the microdroplet and            incubating the microdroplet under conditions effective for            cell lysis;        -   introducing polymerase chain reaction (PCR) reagents and a            plurality PCR primers into the microdroplet, and incubating            the microdroplet under conditions allowing for PCR            amplification to produce PCR amplification products, wherein            the plurality of PCR primers include one or more primers            that each hybridize to one or more oncogenes;        -   introducing a plurality of probes into the microdroplet,            wherein the probes hybridize to one or more mutations of            interest and fluoresce at different wavelengths; and        -   detecting the presence or absence of specific PCR            amplification products by detection of fluorescence of a            probe, wherein detection of fluorescence indicates the            presence of the PCR amplification products;        -   wherein one or more of steps are performed under            microfluidic control.    -   75. The method according to 74, wherein the probes include        TaqMan® probes.    -   76. The method according to 74 or 75, wherein detecting the        presence or absence of specific PCR amplification products by        detection of fluorescence of a probe includes repeatedly imaging        the microdroplet while the microdroplet is subjected to        conditions allowing for PCR amplification to produce PCR        amplification products.    -   77. The method according to 76, including obtaining        time-dependent fluorescence information.    -   78. The method according to any of 74-77, wherein a reagent is        added to the microdroplet by merging the microdroplet with a        second microdroplet including the reagent.    -   79. The method according to any of 74-78, wherein a reagent is        added to the microdroplet using either droplet coalescence or        picoinjection.    -   80. The method according to any of 74-79, wherein a reagent is        added to the microdroplet by a method including:        -   a) emulsifying the reagent into a stream of droplets,            wherein the droplets are smaller than the size of the            microdroplet;        -   b) flowing the droplets together with the microdroplet; and        -   c) merging a droplet with the microdroplet.    -   81. The method according to any of 74-80, wherein a reagent is        added to the microdroplet by a method including:        -   a) jetting the reagent into a fluid jet;        -   b) flowing the fluid jet alongside the microdroplet; and        -   c) merging a droplet with the microdroplet.    -   82. The method according to any of 74-81, wherein a reagent is        added to the microdroplet by a method including using a fluid        injected into the microdroplet as an electrode.    -   83. The method according to any of 74-82, including sorting a        microdroplet.    -   84. The method according to 83, wherein the microdroplet is        sorted based on a property including size, viscosity, mass,        buoyancy, surface tension, electrical conductivity, charge, or        magnetism.    -   85. The method according to any of 74-84, wherein the subject is        mammalian.    -   86. The method according to any of 74-85, wherein the subject is        human.    -   87. The method according to any of 74-86, wherein the subject        has been diagnosed with cancer.    -   88. A method for the detection of cancer in a subject, the        method including:        -   encapsulating in a microdroplet oligonucleotides obtained            from a biological sample from the subject, wherein at least            one oligonucleotide is present in the microdroplet;        -   introducing polymerase chain reaction (PCR) reagents, a            detection component, and a plurality of PCR primers into the            microdroplet and incubating the microdroplet under            conditions allowing for PCR amplification to produce PCR            amplification products, wherein the plurality of PCR primers            include one or more primers that each hybridize to one or            more oncogenes;        -   detecting the presence or absence of the PCR amplification            products by detection of the detection component, wherein            detection of the detection component indicates the presence            of the PCR amplification products; and        -   diagnosing the subject as having cancer or not based at            least in part on the presence or absence of the PCR            amplification products;        -   wherein one or more steps are performed under microfluidic            control.    -   89. The method according to 88, wherein the plurality of PCR        primers includes 10 or more primers.    -   90. The method according to any of 88-89, wherein the plurality        of PCR primers includes primers for 10 or more oncogenes.    -   91. The method according to any of 88-90, further including        introducing a probe into the microdroplet.    -   92. The method according to 91, wherein the probe is introduced        prior to incubating the microdroplet under conditions allowing        for PCR amplification.    -   93. The method according to 91 or 92, wherein the probe is a        TaqMan® probe.    -   94. The method according to any of 88-93, wherein a reagent is        added to the microdroplet by merging the microdroplet with a        second microdroplet including the reagent.    -   95. The method according to any of 88-94, wherein a reagent is        added to the microdroplet using either droplet coalescence or        picoinjection.    -   96. The method according to any of 88-95, wherein a reagent is        added to the microdroplet by a method including:        -   a) emulsifying the reagent into a stream of droplets,            wherein the droplets are smaller than the size of the            microdroplet;        -   b) flowing the droplets together with the microdroplet; and        -   c) merging a droplet with the microdroplet.    -   97. The method according to any of 88-96, wherein a reagent is        added to the microdroplet by a method including:        -   a) jetting the reagent into a fluid jet;        -   b) flowing the fluid jet alongside the microdroplet; and        -   c) merging a droplet with the microdroplet.    -   98. The method according to any of 88-97, wherein a reagent is        added to the microdroplet by a method including using a fluid        injected into the microdroplet as an electrode.    -   99. The method according to any of 88-98, wherein the detection        component is detected based on a change in fluorescence.    -   100. The method according to any of 88-99, wherein detecting the        presence or absence of the PCR amplification products includes        repeatedly imaging the microdroplet.    -   101. The method according to 100, wherein the microdroplet is        repeatedly imaged while the microdroplet is subjected to        conditions allowing for PCR amplification to produce the PCR        amplification products.    -   102. The method according to any of 88-101, including sorting a        microdroplet.    -   103. The method according to 102, wherein the microdroplet is        sorted based on a property including size, viscosity, mass,        buoyancy, surface tension, electrical conductivity, charge, or        magnetism.    -   104. The method according to any of 88-103, including sorting        based at least in part based upon the detection of the presence        or absence of PCR amplification products.    -   105. The method according to any of 88-104, further including:        -   injecting a diluent into the microdroplet; and        -   flowing the microdroplet through a microfluidic channel on            which an electric field is being applied, under conditions            in which the microdroplet is split.    -   106. The method according to any of 88-105, wherein the subject        is mammalian.    -   107. The method according to any of 88-106, wherein the subject        is human.    -   108. The method according to any of 88-107, wherein the subject        has been diagnosed with cancer.    -   109. A microfluidic device including:        -   a cell encapsulation device for encapsulating a cell            obtained from a subject's blood sample in a microdroplet;        -   a first chamber in fluidic communication with the cell            encapsulation device, the first chamber including a first            reagent injector element for adding a first reagent to the            microdroplet, and a heating element;        -   a second chamber in fluidic communication with the first            chamber, the second chamber including a second reagent            injector element for adding a second reagent to the            microdroplet, and a heating element, wherein the heating            element is configured to heat the microdroplet at two or            more temperatures; and        -   a detection region, in fluidic communication with the second            chamber, which detects the presence or absence of reaction            products from the second chamber.    -   110. The microfluidic device as set forth in 109, wherein the        heating element of the second chamber includes a Peltier plate,        heat sink, and control computer.    -   111. The microfluidic device as set forth in 109, wherein the        microfluidic device includes one or more liquid electrodes.    -   112. A single cell RT-PCR microfluidic device including:        -   an input microchannel coupled to a drop maker for            introducing microdroplets into the microfluidic device;        -   a pairing microchannel in fluidic communication with the            input microchannel;        -   a dilution buffer drop maker in fluidic communication with            the pairing microchannel, for producing drops of dilution            buffer that are larger in volume than the microdroplets and            for pairing a single drop of dilution buffer with a single            microdroplet;        -   a merging microchannel in fluidic communication with the            pairing microchannel, for accepting a paired drop of            dilution buffer and microdroplet from the pairing            microchannel;        -   a first electric field generator positioned along the            merging microchannel for producing an electric field that is            capable of merging a paired drop of dilution buffer and            microdroplet in the merging microchannel to form a diluted            microdroplet;        -   a mixing microchannel in fluidic communication with the            merging microchannel, for receiving the diluted microdroplet            from the merging channel and mixing the contents of the            diluted microdroplet;        -   a drop sampler in fluidic communication with the mixing            microchannel, for extracting a sample of the diluted            microdroplet,        -   a picoinjection microchannel in fluidic communication with            the drop sampler, wherein the picoinjection microchannel            includes a picoinjector and is for receiving the sample of            the diluted microdroplet and picoinjecting RT-PCR reagents            into the sample;        -   a second electric field generator, wherein the second            electric field generator is positioned along the            picoinjection microchannel to create an electric field            sufficient to allow for the picoinjection of the RT-PCR            reagents into the sample;        -   a thermocycler heating element in fluidic communication with            the picoinjection microchannel for carrying out an RT-PCR            reaction on the sample picoinjected with the RT-PCR            reagents.    -   113. The microfluidic device of 112, further including an        encapsulating chamber in fluidic communication with the input        microchannel, for encapsulating a cell and lysis reagent into a        microdroplet.    -   114. The microfluidic device of 112, wherein the first and/or        second electric field generators are liquid electrodes connected        to a power supply or high voltage amplifier.    -   115. The microfluidic device of 112, including ridges in one or        more walls of a microfluidic flow channel downstream of the        input microchannel, wherein the ridges are configured to trap a        layer of oil and prevent wetting of the one or more walls of the        flow channel.    -   116. The microfluidic device of 112, including ridges in one or        more walls of a microfluidic flow channel downstream of the        pairing microchannel, wherein the ridges are configured to trap        a layer of oil and prevent wetting of the one or more walls of        the flow channel.    -   117. The microfluidic device of 112, including ridges in one or        more walls of a microfluidic flow channel downstream of the        picoinjection microchannel, wherein the ridges are configured to        trap a layer of oil and prevent wetting of the one or more walls        of the flow channel.    -   118. The microfluidic device of 112, wherein the picoinjection        microchannel is configured to receive a sample that has        undergone an RT-PCR reaction in the sampler and picoinject the        sample with PCR reagents.    -   119. The microfluidic device of 118, wherein the thermocycler is        configured for performing a PCR reaction on a sample        picoinjected with the PCR reagents.    -   120. The microfluidic device of 118, wherein the PCR reagents        and the RT-PCR reagents include the same primers.    -   121. The microfluidic device of 118, wherein the PCR reagents        and the RT-PCR reagents include different primers.    -   122. The microfluidic device of 112, wherein the RT-PCR reagents        includes a bead conjugated with a fluorescent dye and a nucleic        acid probe.    -   123. The microfluidic device of 112, wherein the RT-PCR reagents        includes a fluorescent DNA probe.    -   124. A single cell RT-PCR microfluidic device including:        -   an input microchannel coupled to a flow focus drop maker for            introducing microdroplets into the microfluidic device,            wherein the flow focus drop maker spaces the microdroplets            in the input microchannel by a volume of oil and wherein            each microdroplet including a cell lysate sample;        -   a pairing microchannel in fluidic communication with the            input microchannel;        -   a dilution buffer drop maker in fluidic communication with            the pairing microchannel, for producing a drop of dilution            buffer that is larger in volume than a microdroplet and for            pairing a single drop of dilution buffer with a single            microdroplet;        -   a merging microchannel in fluidic communication with the            pairing microchannel, for accepting a paired drop of            dilution buffer and microdroplet from the pairing            microchannel;        -   a first electric field generator positioned along the            merging microchannel for producing an electric field across            the merging channel that is capable of merging a paired drop            of dilution buffer and microdroplet in the merging            microchannel to form a diluted microdroplet;        -   a mixing microchannel in fluidic communication with the            merging microchannel, for receiving the diluted microdroplet            from the merging channel and mixing the contents of the            diluted microdroplet;        -   a drop sampler in fluidic communication with the mixing            microchannel, for extracting a sample of the diluted            microdroplet,        -   a picoinjection microchannel in fluidic communication with            the drop sampler, wherein the picoinjection microchannel            includes a picoinjector and is for receiving the sample of            the diluted microdroplet and picoinjecting RT-PCR reagents            into the sample;        -   a second electric field generator, wherein the second            electric field generator is positioned along the            picoinjection microchannel to create an electric field            across the picoinjection microchannel sufficient to allow            for the picoinjection of the RT-PCR reagents into the            sample;        -   a thermocycler heating element in fluidic communication with            the picoinjection microchannel for carrying out an RT-PCR            reaction on the sample picoinjected with the RT-PCR            reagents.    -   125. The microfluidic device of 124, further including an        encapsulating chamber in fluidic communication with the input        microchannel, for encapsulating a cell and lysis reagent into a        microdroplet.    -   126. The microfluidic device of 124, wherein the first and/or        second electric field generators are liquid electrodes connected        to a power supply or high voltage amplifier.    -   127. The microfluidic device of 124, including ridges in one or        more walls of a microfluidic flow channel downstream of the        input microchannel, wherein the ridges are configured to trap a        layer of oil and prevent wetting of the one or more walls of the        flow channel.    -   128. The microfluidic device of 124, including ridges in one or        more walls of a microfluidic flow channel downstream of the        pairing microchannel, wherein the ridges are configured to trap        a layer of oil and prevent wetting of the one or more walls of        the flow channel.    -   129. The microfluidic device of 124, including ridges in one or        more walls of a microfluidic flow channel downstream of the        picoinjection microchannel, wherein the ridges are configured to        trap a layer of oil and prevent wetting of the one or more walls        of the flow channel.    -   130. The microfluidic device of 124, wherein the picoinjection        microchannel is configured to receive a sample that has        undergone an RT-PCR reaction in the sampler and picoinject the        sample with PCR reagents.    -   131. The microfluidic device of 130, wherein the thermocycler is        configured for performing a PCR reaction on a sample        picoinjected with the PCR reagents.    -   132. The microfluidic device of 130, wherein the PCR reagents        and the RT-PCR reagents include the same primers.    -   133. The microfluidic device of 130, wherein the PCR reagents        and the RT-PCR reagents include different primers.    -   134. The microfluidic device of 124, wherein the RT-PCR reagents        includes a bead conjugated with a fluorescent dye and a nucleic        acid probe.    -   135. The microfluidic device of 124, wherein the RT-PCR reagents        includes a fluorescent DNA probe.    -   136. The method of any one of 1-20, wherein a reagent is added        to the microdroplet by:        -   contacting the microdroplet with oil so that the oil            encapsulates the microdroplet to form a double emulsion;        -   contacting the double emulsion with a drop containing the            reagent so that the drop containing the reagent encapsulates            the double emulsion to form a triple emulsion;        -   applying an electrical field to the triple emulsion so that            the fluid interfaces of the triple emulsion are ruptured and            allow the microdroplet and reagent to mix.    -   137. The method of 136, wherein the electric field is applied by        one or more liquid electrodes.    -   138. A microfluidic device including: a flow channel, a        microfluidic junction fluidically connected to the flow channel,        and ridges in one or more walls of the microfluidic flow channel        immediately downstream of the microfluidic junction.    -   139. The microfluidic device of 138, wherein the ridges trap a        layer of oil and prevent wetting of the one or more walls of the        flow channel.    -   140. The microfluidic device of 138, wherein the base of each of        the one or more ridges is from about 10 microns to about 20        microns in length.    -   141. The microfluidic device of 138, wherein, the peak of each        of the one or more ridges has a width of about 1 to about 10        microns.    -   142. The microfluidic device of 138, wherein the height of each        of the one or more ridges is from about 5 microns to about 15        microns.    -   143. The microfluidic device of 138, wherein the ratio of the        base of each of the one or more ridges to the height of each of        the one or more ridges is from about 1.0:0.75 to about 0.75:1.0.    -   144. The microfluidic device of 138, wherein the base of each of        the one or more ridges to the height of each of the one or more        ridges to the width of the peak of the one or more ridges is        about 1:0.75:0.5.    -   145. The microfluidic device of 138, wherein the ridges extend        for a distance along the channel wall of from about 50 microns        to about 500 microns.    -   146. The microfluidic device of 138, wherein the ridges extend        for a distance along the channel wall, wherein the ratio between        the distance along the channel wall and the width of the channel        is from about 10:1 to about 1:2.

EXAMPLES

As can be appreciated from the disclosure provided above, the presentdisclosure has a wide variety of applications. Accordingly, thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Those of skill in the art will readily recognizea variety of noncritical parameters that could be changed or modified toyield essentially similar results. Thus, the following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the presentinvention, and are not intended to limit the scope of what the inventorsregard as their invention nor are they intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (e.g.amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for.

Example 1: Microfluidic System for Performing Single-Cell PCR Reactions

Device Manufacturing:

The chips were made using the same photolithographic processes inpolydimethylsiloxane as the other devices described above. A generalschematic of the chips is shown in FIG. 1. The general approach carriedout by such chips is depicted in FIG. 6.

Sample Preparation:

5-25 mL whole blood samples were extracted from a subject via syringe.Nucleated cells were separated using on-chip pinched-flow fractionation,as generally described in Lab on a Chip, 2005, 5, 778-784; thedisclosure of which is incorporated herein by reference. Nucleated cellswere collected for subsequent analysis.

PCR Reactions:

The assay requires the execution of an RT-PCR reaction in dropscontaining concentrated cell lysates; however, cell lysates inhibitRT-PCR (FIG. 7). To overcome this inhibition, a protocol has beendeveloped that utilizes proteinase K to digest inhibitory proteins incell lysates. Using proteinase K allows efficient amplification inlysates at concentrations as high as 1 cell in 50 pL, with optimalamplification occurring at 1 cell in 200 pL (FIG. 7). Thus, the systemoperates at this concentration.

Cell encapsulation, lysis, and proteinase K digestion are accomplishedusing an integrated microfluidic system (FIG. 8, Panels 1-3). Cells areco-encapsulated in 70 μm drops (200 pL) with lysis buffer containingnon-ionic detergents and proteinase K using a 30×30 μm flow focusdevice. Importantly, the cells are not exposed to lysis buffer untilthey are encapsulated in drops, ensuring that no lysis occurs prior toencapsulation. This is enabled by the laminar flow conditions in themicrofluidic channels, which ensure that diffusive mixing is negligiblecompared to the convection of the fluids. Following encapsulation, theclose-packed drops move through a 55° C. incubation channel for 20 min,to allow the cells to lyse and the proteinase K to digest inhibitoryproteins. The drops are then split into equally-sized drops using ahierarchical splitter (FIG. 5; FIG. 8, Panel 3), producing drops of theideal small size for picoinjection and Megadroplet Array imaging (FIGS.12-13).

Prior to injection of the RT-PCR reagents and enzymes, the proteinase Kis inactivated by heating the drops to 95° C. for 10 min. The drops arethen injected with an equal volume of 2× primers and RT-PCR reagents(FIG. 9, Panel A). After picoinjection, the emulsion is collected into aPCR tube and thermal cycled. To determine whether a drop contains acancer cell, TaqMan® probes are also included that hybridize to theEpCAM amplicons; this allows the probes to be hydrolyzed by the 5′-3′nuclease activity of Taq DNA polymerase, liberating the 5′ fluorophorefrom the quenching 3′ end modification making the drop fluorescent. Bycontrast, drops not containing cancer cells do not have EpCAM amplicons,so that the TaqMan® probes remain quenched and non-fluorescent (FIG. 4,Panels A-B). Hence, a bright drop relates the presence of an EpCAMpositive cancer cell (FIG. 9, Panels B-C; FIG. 10). The thermocycleddrops are injected into a flow cell 30 μm in height and 54 cm² in area;the narrow vertical gap of the flow cell forces the emulsion into amonolayer, allowing unobstructed epi-fluorescence visualization of everydrop. For the fluorescence imaging, an automated microscope captures amosaic of the entire flow cell and stores the images on a hard drive.The images are processed with custom Matlab code to identify fluorescentdrops and measure their brightness. All data is stored digitally andanalyzed using custom algorithms.

Example 2: Quantitative Multiplexed Assay

To screen more than one gene simultaneously, a multiplexed qPCR reactionmay be utilized. Reactions were initially performed in bulk with PCRtubes to optimize reaction conditions. Using these methods, successfulmultiplexing was achieved during digital droplet RT-PCR for threeTaqMan® probes, EpCAM, CD44 and CD45. An example of this multiplexing isshown in FIG. 11, where EpCAM and CD44 probes were multiplexed in dropscontaining both target transcripts. All PCR primer sets were designed tospan large introns, making these larger genomic PCR products highlyunlikely in multiplex reactions. Additionally, all TaqMan® probes aredesigned to hybridize to exon-exon junctions. The current probe sets donot recognize gDNA.

Single-Cell qPCR with Megadroplet Arrays:

To perform qPCR analysis on single cells, the drops are imaged as theyare thermal cycled. This requires that the drops be held at fixedpositions during thermal cycling so they can be repeatedly imaged. Themicrofluidic system used to prepare the drops was prepared as describedabove and in Example 1. After the drops are formed and loaded with cellsand qPCR reagents, they are introduced into a Megadroplet Array (FIG.12, Panels A-C; FIG. 13). The array consists of channels in which thechannel ceilings are indented with millions of circular traps 25 μm indiameter. When the drops flow into the array, they are slightly pancakedin shape because the vertical height of the flow channel is 15 μm, or 10mm shorter than the drops. When a drop nears a trap, its interfaceadopts a larger, more energetically favorable radius of curvature. Tominimize its surface energy, the drop will entirely fill the trap,allowing it to adopt the lowest, most energetically favorable, averageradius of curvature. The capillary pressure of the drop is severalorders of magnitude larger than the shear exerted by the flow, ensuringthat the drops remain intact and confined in the traps. After a trap isoccupied by a drop, no other drops are able to enter because the trapwill be large enough to fit only one drop; additional drops are diverteddownstream, to occupy the first vacant trap they encounter. The array isfilled using a close-packed emulsion, and thus every trap is occupied bya drop. After the droplet array is filled, oil is injected to removeexcess drops and the array is thermal cycled and imaged.

Thermal System for Temperature Cycling and Imaging:

Once the array is filled with drops and cells, the device is thermalcycled while simultaneously imaging the drops, to obtain thetime-dependent information necessary for qPCR. The thermal cycling isaccomplished using a custom heater consisting of a Peltier plate, heatsink, and control computer (FIG. 13). The Peltier plate permits heatingor cooling the chip above or below room temperature by controlling theapplied current. To ensure controlled and reproducible temperature, acomputer monitors the temperature of the array using integratedtemperature probes, and adjusts the applied current to heat and cool asneeded. A copper-plate allows uniform application of heat anddissipation of excess heat during cooling cycles, enabling cooling from95° C. to 60° C. in under 1 min execution of the qPCR assay in under twohours. To image the droplets during temperature cycling, a customizedCanoscan 9000F scanner bed having a resolution of 9600 dpi by 9600 dpiis utilized. For 10 million hexagonally-packed 25 μm drops (54 cm²), 800million pixels are required at highest resolution. With a resolution of20 pixels per drop, the full image may be captured in 3 s. The array isimaged several times per cycle with different excitation and emissionfilters to visualize the different dyes for the multiplexed TaqMan®probes.

Example 3: Electrode-Free Picoinjection of Drops of Microfluidic Drops

Microfluidic devices were fabricated in poly(dimethylsiloxane) (PDMS)using soft photolithographic techniques. The devices had channel heightsof 30 μm, optimal for the picoinjection of water-in-oil droplets thatare 50 μm in diameter. The device design is similar to those describedpreviously by Abate, et al. Proc. Natl. Acad. Sci. U.S.A., 2010, 107,19163; the disclosure of which is incorporated herein by reference. Animportant difference, however, is that the channels for the metal solderelectrodes are removed. Further, a “Faraday Mote”—an empty channelfilled with a conducting aqueous solution—is implemented that runsbetween the injection site and the droplet spacer, as shown in FIG. 15,Panel B. The mote electrically isolates re-injected drops upstream ofthe picoinjection site from electric fields emanating from thepicoinjector, preventing unintended merging. The emulsion that waspicoinjected consists of monodisperse droplets of 3.8 mM fluoresceinsodium salt (C₂₀H₁₀Na₂O₅) dissolved in Milli-Q H₂O. The droplets aresuspended in a carrier oil of Novec HFE-7500 fluorinated oil with 2%(wt/wt) dissolved biocompatible surfactant. The picoinjection fluidsconsist of a dilution series of NaCl ranging from 0 to 500 mM, eachcontaining 3.8 mM fluorescein sodium salt. This range of concentrationsreflects the molarities of dissolved ions present in most biologicalbuffers and reagents. Thus, since in most applications the fluids willalready contain the requisite ions, the technique can be used withoutadding additional reagents to the solutions.

Droplets and carrier oil were introduced via syringe pumps (New Era) andspaced using the same carrier oil and surfactant mixture described above(FIG. 15, Panels A-B). The picoinjection fluid was contained in a BDFalcon tube. Through the cap of the Falcon tube was submerged a wireelectrode into the fluid, as illustrated in FIG. 15, Panel A. Gaps inthe cap were sealed with LocTite UV-cured epoxy. The picoinjection fluidwas charged using a function generator outputting a 10 kHz sinusoidalsignal ranging from 0 to 5 volts. This output was amplified 1000× by aTrek 609E-6 model HV amplifier. The positive output of the amplifier wasattached via an alligator clip to the wire submerged in thepicoinjection fluid. The ground electrode of the amplifier was attachedto the metal needle of a syringe containing a 1 M solution of NaCl,introduced into the Faraday Mote (FIG. 15, Panel A). The two electrodeswere never in electrical contact and the emulsions exiting the devicewere collected into separate, electrically isolated containers to avoida closed circuit and prevent current flow.

The picoinjected reagent was infused into the device through PE-2 tubing(Scientific Commodities) using an air pressure pump (ControlAir Inc.)controlled by custom LabVIEW software. The injection fluid waspressurized such that the oil/water interface at the picoinjectionorifice is in mechanical equilibrium with the droplet channel; thepressure difference across the interface is equal to the Laplacepressure, causing the injection fluid to bulge into the droplet channelwithout budding off and forming its own drops (FIG. 15, Panel C). Forthis device, drops and spacer oil were injected the at flow rates of 200and 400 μl hr⁻¹, respectively. At these flow rates, the picoinjectionfluid interface is in mechanical equilibrium for an applied pressure of˜13 psi. The lengths of the tubing carrying the injection fluid andsolution serving as a Faraday mote was controlled, since longer tubeshave higher electrical resistance and may attenuate the AC signalapplied to trigger picoinjection.

To picoinject drops with reagent, the previously formed monodisperseemulsion was re-injected into the picoinjection device. The emulsion wasintroduced at a high volume-fraction such that there is little carrieroil and the drops are packed together. The packed drops traveled througha narrowing channel that forced them single file. Additional oil withsurfactant is added from two perpendicular channels, spacing the dropsevenly, as shown in FIG. 15, Panel B. A simple T-junction spacer wasalso found to work. The droplets then passed the picoinjector, a narrowchannel containing the reagent to be added. To trigger picoinjection,the voltage signal was applied to the electrode submerged in theinjection fluid, generating an electric field at the picoinjector as thedrops pass the injection site. This caused the drops to coalesce withthe injection fluid. As they traveled past, fluid was injected into themthrough a liquid bridge formed after the two fluids coalesce. Theapplied signal must have zero offset to prevent electrophoreticmigration of charged particles in the solutions. Additionally, thefrequency of the signal must be high enough to ensure that during theact of injecting, the sign of the field switches many times betweenpositive and negative, so that the net charge of the fluid added to thedroplets is approximately zero. This ensured that the droplets leavingthe injector have zero net charge, which was important for ensuring thatthey remain stable. A 10 kHz signal was applied.

To analyze the behavior of the picoinjector, the injection site wasobserved under a microscope. In the absence of an electric field, adistinct boundary was observed between the droplet and the injectionfluid, as shown in FIG. 16, Panel A. When a 250 V signal was applied tothe picoinjector, the boundary vanishes and droplet coalescence isvisible, as demonstrated in FIG. 16, Panel B. Thus, electrification ofthe injection fluid is adequate to trigger picoinjection, demonstratingthat electrically-isolated electrodes are not needed.

To determine if it were possible to vary the injection volume using theapplied voltage, voltage was varied between 0-5000V and the volumechange of the resulting droplets was measured. Injection volume wasquantified with an optical fluorescence detection setup. As the dropspassed a 472 nm wavelength laser focused on the droplet channel˜1 cmdownstream of the picoinjector, the emitted fluorescence signal from thedissolved fluorescein contained within the drops was amplified by aphotomultiplier tube (PMT) and converted to a voltage signal analyzedwith LabVIEW FPGA. As the drops passed the laser, their fluorescencesignals resembled square waves as a function of time, with amplitudesand widths that corresponded to the drop intensity and length,respectively. The drops had a spherical diameter larger than thedimensions of the channel, causing them to be cylindrical in shape.Thus, the drop volume is approximately linear as a function of length.To calculate the volume fractional (Vf) increase, the ratio of the droplength before and after picoinjection was measured. These measurementswere repeated for a range of applied voltages and molarities of NaCl inthe injection fluid.

The increase in volume was plotted as a function of applied voltage forthree representative molarities of injection fluid in FIG. 17, PanelsA-C. In all cases the injection volume increased with the appliedvoltage, though this effect is most prominent for the 100 mM injectionsolution shown in FIG. 17, Panel A. The dependence of the droplet volumeon the applied voltage may be attributed to the observation that thedroplets are not perfect cylinders as they travel past the picoinjector;instead they have a “bullet” shape, with the leading edge having asmaller radius of curvature than the trailing edge. Consequently, as thedrops pass the picoinjector, the thickness of the oil layer separatingtheir interface from the bulge of the picoinjection fluid decreases. Foran electrically-induced thin-film instability, the threshold voltagerequired to rupture the interface depends on the thickness of the film,decreasing as the film gets thinner. Hence, because the film thicknessdecreases as the drops pass the picoinjector, the moment of coalescencedepends on electric field magnitude: for higher fields it is possible torupture thicker films, leading to picoinjection at an earlier point;conversely, for lower fields thinner films are ruptured, causingpicoinjection to start at a later point. Because the volume injecteddepends on the duration of picoinjection, it therefore also depends onapplied voltage. This is supported by data which shows a dependence onapplied voltage for all molarities (FIG. 17, Panels A-C). It was alsoobserved that the curves relating volume injected to applied voltage arelower for lower molarities, as shown for the 50 mM and 25 mM data inFIG. 17, Panels B and C, respectively. This may be attributable to thefact that lower molarity solutions have a lower conductivity, and canthus attenuate the AC signals used to trigger injection, reducing thevolume injected for a particular applied voltage.

Above 3000V and 100 mM, the injected volume begins to decrease and thevariability in drop size increases. In images of these systems at thesevoltages, it was observed that the picoinjection fluid is no longer heldat equilibrium in the picoinjection orifice, but instead wets thechannel walls and buds off small drops into the flow channel.

To characterize the behavior of the electrode-free picoinjector for allparameters, injection volume was measured as a function of molarity andapplied voltage and the resulting data was plotted on a 2D heat-map(FIG. 18). This data demonstrates that the technique should allowcontrolled picoinjection for most biological buffers, which commonlyhave molarities within the tested range.

To investigate whether the electric fields and currents generated by thehigh-voltage signal may disrupt biomolecules needed for downstreamassays, the picoinjector was used to prepare droplets for an RT-PCRreaction. Drops containing total RNA isolated from an MCF7 human cellline were picoinjected with an RT-PCR reaction mixture containing theenzymes reverse transcriptase (RT) and Taq DNA polymerase.Negative-control drops were injected with a mixture containing noenzymes. Additional non-emulsified positive and negative controlreactions were performed in parallel with the same RT-PCR mixture.Following thermocycling, the emulsions were broken and the amplificationproducts visualized on an ethidium bromide-stained 2% agarose gel. Thepositive control and picoinjected drops showed PCR bands of comparableintensity for the expected 100 bp amplicon length, as visible in FIG.19. In contrast, the negative controls showed no amplification,demonstrating that applying the triggering signal to the picoinjectionfluid is sufficiently biocompatible so as to allow downstream RT-PCRreactions in drops.

Example 4: Coalescing Triple-Emulsions to Add Reagent to Droplets

One step, which may be important in running a droplet reaction, is theability to add reagents to pre-existing drops. As an example, dropaddition might be beneficial if a final drop reaction requires a reagentthat could be denatured in a prior heating step. If no drop-stabilizingsurfactants are used, adding reagent can be as simple as bringing a dropin contact with a second reagent-filled one. Standard drop processingand storage often require surfactant-stabilized drops, however, andlocalized electric fields have been utilized to selectively disrupt andmerge pairs of drops. Merging involves timing the flow of original andreagent drops so that they pair up and are in contact. A second strategyuses electric fields to destabilize a passing drop so it can be injectedwith reagent from a side channel. This avoids the issue ofsynchronization, but has the disadvantage that each drop is potentiallycross-contaminated when joined with the side channel. Furthermore, onlya volume less than or equal to the passing drop can be injected.

Rather than merging or injecting reagents with a drop, presented here isa different scheme where the original drop is enveloped within a largerreagent droplet and then both are coalesced via application of anelectric field. In some embodiments, this enveloping facilitates thepairing of one original drop with one reagent envelope. The containednature of the mixing may also limit cross-contamination and facilitatethe addition of arbitrary volumes as compared with a droplet injector.

The drop-envelope pairing is made possible with surface chemistry. Toreduce interfacial energy, a hydrophilic channel encapsulates anoil-coated drop in aqueous reagent if available. A subsequenthydrophobic channel then encapsulates it in oil, creating a stablewater-in-oil drop in a water-in oil drop, or triple emulsion (E3). Thistechnique of alternating channel hydrophobicity has each low-orderemulsion triggering the formation of the next higher one, with reliablequintuple emulsions even possible. The triggering leads to the properpairing of one original drop per envelope. Once there, the original dropsurface is in maximal contact with the inner surface of the reagentenvelope, facilitating later electro-coalescence. This contact meansthat any volume of reagent could be added to the original drop, from athin-shelled reagent envelope of fractional volume to an envelope 10²,10³, 10⁴ or more times larger.

A detailed schematic of the E3 scheme is shown in FIG. 23. First, apremade, water-in-oil emulsion (E1) was reinjected into the devicethrough a hydrophilic channel (FIG. 23, top left). The drops met ajunction where co-flowing reagent pinched them off individually,surrounding them to reduce surface repulsion. The oil of the E1 formedthin, stable shells that housed each original drop. The channelimmediately after the junction was designed to include ridges asdescribed herein to traps pockets of aqueous fluid. This prevented oilfrom contacting the walls during budding and potentially altering theirhydrophobicity. The water-in-oil-in water double emulsion (E2) thentraveled to a second junction where it met a hydrophobic channelcarrying oil (FIG. 23, bottom left) (Additional description andcharacterization of double emulsions and their formation are provided inthe descriptions of FIGS. 38-51). Here, the aqueous reagents wererepelled from the walls, and formed an E3 drop. In the figure, the E2 isshown in the process of seeding the E3 by weakening the adhesion of thereagent fluid to the hydrophilic channel. The volume ratio of reagent tothe original E1 drops was determined by the flow rates at the firstjunction.

After formation, the E3 was passed into a narrow constriction andcoalesced with an electric field. The electric field was generatedbetween two salt-solution containing channels, an electrode carrying ahigh, alternating voltage and a grounded moat (FIG. 23, bottom). Theconstriction may have facilitated application of the electric field tothe drops because the reagent envelope likely contained mobile ions thatcould screen the interior from the electric field. As seen in thefigure, constricting the E3 forces the inner drop to the channel wall.After coalescing, the oil shell collapsed and became the innermost phaseof an inverted oil-water-oil double emulsion (E2′).

The device itself was constructed using conventional PDMS fabricationtechniques. First, a master was made by spinning layers of SU-8 resistonto a silicon wafer and sequentially exposing them with UV light(Blakray) and a patterned mylar mask (Fineline Imaging). Afterdeveloping in CD-30, the SU-8 master was covered in PDMS (PDMSmanufacturer) with a 10:1 polymer to cross-linker mix, placed in vacuumto remove trapped air, and baked for 1 hour at 75° C. The device wasthen extricated and given access holes with a 0.75 mm biopsy punch.Next, the device was bonded to a 1 mm-thick glass slide by exposing bothto 1 mbar O₂ in a 300 W plasma cleaner for 20 s, attaching, and thenbaking for 10 min at 75° C.

The final processing steps created the hydrophilic and hydrophobicchannels. First, Aquapel® was flowed backwards through the device, intothe drop outlet and out the carrier oil inlet. At the same time, thedrop reinjector inlet was pressurized with 15 psi air to prevent theAquapel® from entering the double-emulsion, hydrophilic section of thedevice. Next, the same inlets exposed to Aquapel® were plugged with PEEKtubing (Resolution Systems, TPK.515-5M) and the device was re-exposed to1 mbar O₂ plasma in the same cleaner for 1 min. The plasma made exposedchannels hydrophilic, while the plugs kept the hydrophobic channels asthey were. This hydrophilic treatment was only semi-permanent, and othermethods not used here are capable of creating robust hydrophilicchannels.

To operate, syringes filled with the appropriate fluids were connectedto the finished device via PE-2 tubing (Scientific Commodities,#BB31695) and the same PEEK tubing and pressurized using syringe pumps(New Era). The reinjected drops consisted of Milli-Q water in afluorinated oil (Novec HFE 7500) with a 1% w/w biocompatible surfactant.The drops were flowed at a relatively slow flow rate of 20 μL/hr, and asnaking channel was used (FIG. 23, top left) to add flow resistance andfilter any pressure fluctuations. The test reagent was PBS buffer(model#) with 0.1% pluronic surfactant (model #), and the carrier oilwas the same as with the reinjected drops. These were flowed at equalrates between 200 μL/hr and 1200 μL/hr. The electrodes and moat werefilled with 3.0 NaCl solution. The electrode, which was a dead end, waspressurized with a solution-filled syringe until air in the channel wasabsorbed by the PDMS. It was connected to a 20 kHz high voltageoscillator (JKL Components Corp, BXA-12579) running at 500 V. Such largevoltages applied to merge or inject drops have been shown to bebiologically compatible.

FIG. 24 shows microscope images of the running E3 device. The reinjectedE1 travelling from the top of FIG. 24, Panel A, are starkly outlinedbecause the disparate oil and water indices of refraction bent the backlighting. After the E1 was encapsulated at the junction by reagentflowing from the sides and became an E2, the inner and outer indices ofrefraction matched and the borders became much fainter. This is anindication of the thinness of the oil shell, which did not appreciablyrefract. In FIG. 24, Panel A, the E1 consisted of 30 μm-diameter drops(15 pL), and all channels here were hydrophilic and square, 30 μm to aside.

At the next junction, seen in FIG. 24, Panel B, the E2 exited thehydrophilic channel as an E3 in a large square, hydrophobic channel, 60μm to a side. As with the initial emulsion, the edges of these E3 dropswere clearly visible due to refractive mismatch. Conceivably, this stepcould have caused timing issues because the inner E1 needed tosynchronize with the large drop formation. However, this problem wasavoided because the arrival of the E1 at the junction weakened theadhesion of the reagent phase to the hydrophilic channel and inducedbudding. The process is shown in the inset of FIG. 24, Panel B, andcaused a very regular loading of E1 into the E3.

The coalescence of the E3 is shown in FIG. 24, Panel C. The 60 μm-widechannel narrowed to 15 μm, squeezing the E1 against the walls where theelectric field from the electrode could penetrate. The new E2′ productof coalescing can be seen on the right. The collapsed oil remnantsappear in high contrast and have a volume of roughly 2 pL, correspondingto an original oil shell that was 1 μm thick. The remnants couldconceivably have merged with the carrier oil during coalescence exceptfor the fact that the E3 was squeezed against the channel wall wherethere is no oil. In the inset, the constriction is shown withoutelectric field. No coalescence occurred and the constriction moved theinner phases to the rear. The regularity of coalescence is demonstratedin FIG. 24, Panel D, the top of which shows a mixing channel forhomogenizing the aqueous contents of the drop.

The precise dynamics of E3 coalescing were determined using a fastcamera. Two time series are shown in FIG. 25, with the oil shell of theinner E1 highlighted in blue (indicated by arrows in FIG. 25). Eachstarts out at a time t=−0.7 ms where the inner E1 was not yetconstricted and was spherical. Time t=0.0 ms was set immediately beforerupturing when the E1 was pinned against the constriction walls andslightly flattened. By next frame, t=0.1 ms, the E1 ruptured. In FIG.25, Panel A, the rupturing ejected contents of the E1 to the back of thedrop, whereas in FIG. 25, Panel B, the contents were ejected forward. Inhigh-order emulsions, the unconstrained surface of an inner phase willbe tangent somewhere with the surface of the next outermost phase toreduce interfacial energy (i.e. the phases are never perfectlyconcentric). This randomly positioned contact point helps merging andmay determine where the drop ruptures. After rupturing, the oil shellscollapsed as shown in the frame at t=1.1 ms.

The robustness of this process depends on the appropriate channels beinghydrophilic or hydrophobic. If the first section of the device is notsufficiently hydrophilic, the oil of E1 may wet the channel wallsimmediately after the junction. Instead of travelling as spheres downthe center of the channel as in FIG. 24, Panel A, they may travel ashemispheres down the side and slip into the carrier fluid at the nextjunction as a single emulsion rather than enveloped. If the secondsection of the device is not sufficiently hydrophobic, there may beelectro-wetting at the constriction and small satellite drops will buffoff at the tail of the passing E3. As is, this scheme produces aqueousdrops with oil in them (E2′) as opposed to the pure aqueous drops (E1)of the merger and injector strategies mentioned previously. Depending onthe desired product, this might be acceptable; otherwise, varioustechniques like microfluidic centrifuges or drop splitting can beemployed to remove the oil.

From the study described, a triple emulsion coalescence strategy wasdemonstrated to be a robust method for adding a reagent to a collectionof drops. Such triple emulsion coalescence was carried out without lossof drops or drop mixing, owing to the surface chemistry of the channelsrather than careful synchronization.

Example 5: Picoinjection Enables Digital Detection of RNA Molecules withDroplet RT-PCR

Most biological assays require the stepwise addition of reagents atdifferent times. For microfluidic techniques to be most widely useful, arobust procedure for adding reagents to drops is therefore important.One technique for accomplishing this is electrocoalescence of drops, inwhich the reagent is added by merging the drop with a drop of thereagent using an electric field. Another technique is picoinjection,which injects the reagent directly into the drops by flowing them past apressurized channel and applying an electric field. An advantage ofpicoinjection is that it does not require the synchronization of twostreams of drops, making it easier to implement and more robust inoperation. However, variability in the volume injected from drop to dropand the potential degradation of reagents by the electric field mayinterfere with assays. In addition, during picoinjection, the dropstemporarily merge with the reagent fluid, potentially allowing transferof material between drops, and cross-contamination.

This study investigated the impact of picoinjection on biological assaysperformed in drops and the extent of material transfer between drops.Using sensitive digital RT-PCR assays, it is shown that picoinjection isa robust method for adding reagents to drops, allowing the detection ofRNA transcripts at rates comparable to reactions not incorporatingpicoinjection. It was also determined that there is negligible transferof material between drops. The benefit of workflows incorporatingpicoinjection over those that do not is that picoinjection allowsreagents to be added in a stepwise fashion, opening up new possibilitiesfor applying digital RT-PCR to the analysis of heterogeneous populationsof nucleic acids, viruses, and cells.

Materials and Methods

Microfluidic Device Fabrication

The microfluidic devices consisted of polydimethylsiloxane (PDMS)channels bonded to a glass slide. To make the PDMS mold, a device masterwas first created by spinning a 30 mm-thick layer of photoresist (SU-83025) onto a silicon wafer, followed by a patterned UV exposure andresist development. Next, an uncured mix of polymer and crosslinker(10:1) was poured over the master and baked at 80° C. for 1 hour. Afterpeeling off the cured mold, access holes were punched in the PDMS slabwith a 0.75 mm biopsy coring needle. The device was washed withisopropanol, dried with air, and then bonded to a glass slide followinga 20 s treatment of 1 mbar O₂ plasma in a 300 W plasma cleaner. To makethe devices hydrophobic, the channels were flushed with Aquapel® andbaked at 80° C. for 10 min.

RNA Isolation

Human PC3 prostate cancer or Raji B-lymphocyte cell lines were culturedin appropriate growth medium supplemented with 10% FBS, penicillin andstreptomycin at 37° C. with 5% CO₂. Prior to RNA isolation, Raji cellswere pelleted and washed once in phosphate buffered saline (PBS).Confluent and adhered PC3 cells were first trypsinized prior topelleting and washing. Total RNA was isolated from cell pellets using anRNeasy Mini Kit (Qiagen). Total RNA was quantified using aspectrophotometer and the indicated amounts (between 150 and 1000 ng) ofRNA were used in subsequent 25 ml RT-PCR reactions.

TaqMan® RT-PCR Reactions

The sequence of amplification primers used for the RT-PCR reactions wereas follows: EpCAM Forward 5′-CCTATGCATCTCACCCATCTC-3′ (SEQ ID NO:1),EpCAM Reverse 5′-AGTTGTTGCTGGAATTGTTGTG-3′ (SEQ ID NO:2); CD44 Forward5′-ACGGTTAACAATAGTTATGGTAATTGG-3′ (SEQ ID NO:3), CD44 Reverse5′-CAACACCTCCCAGTATGACAC-3′ (SEQ ID NO:4); PTPRC/CD45 Forward5′-CCATATGTTTGCTTTCCTTCTCC-3′ (SEQ ID NO:5), PTPRC/CD45 Reverse5′-TGGTGACTTTTGGCAGATGA-3′ (SEQ ID NO:6). All PCR primers were validatedprior to use in microfluidic droplet experiments with tube-based RT-PCRreactions. Products from these reactions were run on agarose gels andsingle bands of the predicted amplicon size were observed for eachprimer set. The sequence of the TaqMan® probes was as follows: EpCAM5′-/6-FAM/ATCTCAGCC/ZEN/TTCTCATACTTTGCCATTCTC/IABkFQ/-3′ (SEQ ID NO:7);CD44 5′-/Cy5/TGCTTCAATGCTTCAGCTCCACCT/IAbRQSp/-3′ (SEQ ID NO:8);PTPRC/CD45 5′-/HEX/CCTGGTCTC/ZEN/CATGTTTCAGTTCTGTCA/IABkFQ/-3′ (SEQ IDNO:9). Pre-mixed amplification primers and TaqMan® probes were orderedas a PrimeTime Standard qPCR assay from Integrated DNA Technologies(IDT) and were used at the suggested 1× working concentration.Superscript III reverse transcriptase (Invitrogen) was added directly toPCR reactions to enable first stand cDNA synthesis. Followingemulsification or picoinjection of RT-PCR reagents, drops were collectedin PCR tubes and transferred to a T100 Thermal Cycler (BioRad).Reactions were incubated at 50° C. for 15 min followed by 93° C. for 2min and 41 cycles of: 92° C., 15 s and 60° C., 1 min.

Emulsion Generation and Picoinjection

The reaction mixtures were loaded into 1 mL syringes and injected intomicrofluidic T junction drop makers using syringe pumps (New Era)controlled with custom LabVIEW software. The dimensions of the deviceand flow rates of the reagents were adjusted to obtain the desired 30 mmdrop size. To apply the electric field for picoinjection, the electrodeand surrounding moat channels were filled with a 3M NaCl solution,having a conductivity of ˜0.1 S/cm. The electrode was energized using 20kHz, 300 VAC signals generated by a fluorescent light inverter (JKLComponents Corp) attached via an alligator clip to the syringe needle.

Immunofluorescence Imaging

To image the thermocycled droplets, 10 mL of emulsion were pipetted intoCountess chambered coverglass slides (Invitrogen). The slides wereimaged on a Nikon Eclipse Ti inverted microscope using conventionalwidefield epifluorescence and a 4× objective. Fluorescence filters werechosen to optimize the signal intensity and to mitigate backgroundfluorescence due to spectral overlapping of the dyes used in themultiplexed reactions. The images were captured using NIS Elementsimaging software from Nikon.

Data Analysis

The droplet images were analyzed using custom MATLAB software. For eachfield of view, brightfield and fluorescence images were captured. Thesoftware first located all drops in the brightfield image by fittingcircles to the drop interfaces. Next, the light background in thefluorescence images was subtracted using a smooth polynomial surfaceconstrained to vary over size scales much larger than the drops. Thesoftware then measured the average fluorescence intensity within eachdroplet's circular boundary. The resultant intensity values were offsetso that the cluster of lowest intensity (empty) had an average of zero.Drops were determined to be “positive” or “negative” based on whethertheir intensity fell above or below, respectively, a defined threshold.

Results

Detection of RNA Transcripts in Picoinjected Drops.

A potential concern when using picoinjection for RT-PCR assays is thepossibility that it may interfere with reactions in the drops; forexample, the process may result in variability in the amount of reagentsbetween the drops or degradation of key components upon exposure to theelectric field. To investigate these issues, the detection of twocancer-relevant human transcripts, EpCAM and CD44, was compared inpicoinjected and non-picoinjected drops using TaqMan® RT-PCR, (FIG. 26).The TaqMan® probe for detecting EpCAM was conjugated to the fluorophore6 carboxyfuoroscein (FAM) and the probe for CD44 to the dye Cy5. Theprobe mix also contained primers that flank the TaqMan® probes andyield˜150 base amplicons from these genes.

To prepare the non-picoinjected control drops, the probe mix was addedto a 25 ml RT-PCR master mix reaction containing 150 ng of total RNAisolated from the human PC3 prostate cancer cell line. The RT-PCRsolution was the emulsified into monodisperse 30 mm (14 pL) drops with aT-junction drop maker, and the drops were collected into PCR tubes andthermocycled (FIG. 26, Panel A and 26, Panel C). During thermocycling,drops containing at least one EpCAM or CD44 transcript were amplified,becoming fluorescent at the wavelengths of the associated FAM and Cy5dyes. By contrast, drops without a molecule did not undergoamplification and remained dim, as in standard TaqMan®-based digitaldroplet RT-PCR. Following thermocycling, the drops were pipetted intochambered slides and imaged with a fluorescence microscope. To measurethe concentrations of EpCAM and CD44 in the original solution, thenumber of drops with FAM or Cy5 fluorescence were counted. The reactionsshowed a digital fluorescent signal for both the EpCAM and CD44 probes,indicating that these transcripts were present at limitingconcentrations in the drops, as shown in FIG. 27, Panel A. Controlreactions where reverse transcriptase was omitted failed to produce afluorescent signal, indicating that the TaqMan® assays were specific andnot the result of non-specific cleavage of TaqMan® probes caused by theemulsification process.

To test the impact of picoinjection on TaqMan® RT-PCR, a similarexperiment as above was performed, but the RT-PCR reagents wereseparated into two solutions added at different times. Total RNA, RT-PCRbuffer, primers, probes, and DNA polymerase were emulsified into 30 mmdiameter drops; these drops were not capable of RT-PCR, since theylacked reverse transcriptase. Using picoinjection, an equal volume of 2×reverse transcriptase was introduced in PCR buffer and the drops werethermocycled. Just as with the non-picoinjected control, this emulsionshowed a robust digital signal and had an equivalent ratio offluorescent-to-non-fluorescent drops, as shown in FIG. 27, Panels A andB. To confirm that the fluorescence was not due to background hydrolysisof the TaqMan® probes, disruption of the probes by the electric field,or some other factor, additional reactions were performed where apicoinjection fluid lacking reverse transcriptase was added toRNA-containing drops. In these drops, no fluorescence was evidentfollowing thermocycling (FIG. 27, Panel C), demonstrating that thesignal was indeed a result of digital detection of RNA molecules, andthat these assays were specific.

Quantification of RT-PCR Detection Rates in Picoinjected Drops

To precisely quantify the impact of picoinjection on TaqMan® RT-PCRtranscript detection, four independent replicates of the picoinjectedand non-picoinjected drops were collected. To automate data analysis, acustom MATLAB software was used to locate the drops in the images andmeasure their fluorescence intensities. For a particular channel (FAM orCy5), the fluorescence intensity within each drop was averaged; all dropvalues were subsequently offset so that the cluster of empty drops hadan average of zero (See Materials and Methods). Using one threshold forboth channels, each drop was labeled as positive or negative for EpCAMand CD44 based on whether it was above or below the threshold,respectively, as shown in FIG. 28, Panel A. In total, 16,216 controldrops and 14,254 picoinjected drops were analyzed from the fourexperimental replicates. To determine the TaqMan® detection rate ofpicoinjected drops relative to non-picoinjected controls, the totalnumber of CD44 (Cy5) and EpCAM (FAM) positive control drops in eachreplicate was normalized. Following picoinjection of reversetranscriptase, 92% (+/−26%) of CD44 positive drops and 87% (+/−34%) ofEpCAM positive drops were detected relative to the control drops (FIG.28, Panel B). Although the average transcript detection rate forpicoinjected drops was slightly lower than that of control drops for agiven RNA concentration, the difference was not statisticallysignificant, and some experimental replicates had detection rates forpicoinjected drops higher than for the controls. Based on these results,it was conclude that picoinjection affords transcript detection ratesequivalent to that of digital RT-PCR, with the benefit of allowing thereaction components to be added at different times.

Discrete Populations of Drops can be Picoinjected with MinimalCross-Contamination

An important feature when adding reagents to drops is maintaining theunique contents of each drop and preventing the transfer of materialbetween drops. Unlike the merger of two discrete drops, the contents ofa picoinjected drop become momentarily connected with the fluid beingadded, as illustrated in FIG. 26, Panel B. After the drop disconnectsfrom the fluid, it may leave material behind that, in turn, may be addedto the drops that follow. This could lead to transfer of materialbetween drops, and cross-contamination. To examine the extent to whichpicoinjection results in cross-contamination, TaqMan® RT-PCR reactionswere again used because they are extremely sensitive and capable ofdetecting the transfer of just a single RNA molecule. A FAM-conjugatedTaqMan® probe was used for targeting the EpCAM transcript and ahexachlorofluorescein (HEX) conjugated TaqMan® probe was used forrecognizing the B-lymphocyte-specific transcript PTPRC. Total RNA wasisolated from PC3 cells expressing EpCAM but not PTPRC, and aB-lymphocyte derived cell line (Raji) expressing PTPRC but not EpCAM.For a control set of drops, the RNA from both cell types was mixed,TaqMan® probes and RT-PCR reagents were added, and the solutions wereemulsified into 30 mm drops. The drops were collected into a tube,thermocycled, and imaged, FIG. 29A. In the images, a large number ofdrops displayed FAM and HEX fluorescence, indicative of multiplexedTaqMan® detection of PTPRC and EpCAM transcripts. A smaller fraction hadpure green or red fluorescence, indicating that they originallycontained just one of these molecules, while even fewer were dim andwere thus devoid of these transcripts.

To observe the rate of picoinjector cross-contamination, a microfluidicdevice was used that synchronously produced two populations of dropsfrom opposing T-junctions, pictured in FIG. 29, Panel B. One populationcontained only Raji cell RNA and PTPRC transcripts; the other, only PC3cell RNA and EpCAM transcripts, as illustrated in FIG. 29, Panel B. Bothpopulations contained primers and TaqMan® probes for EpCAM and PTPRC andwere therefore capable of signalling the presence of either transcript.Immediately after formation, the drops were picoinjected with the 2×reverse transcriptase, thereby enabling first strand cDNA templatesynthesis for the TaqMan® assay, and an opportunity for contamination.If RNA was transferred between drops, some of the drops should havedisplayed a multiplexed TaqMan® signal, whereas in the absence ofcontamination, there should have been two distinct populations and nomultiplexing. In the fluorescence images, two distinct populations wereobserved, one positive for EpCAM (FAM) and the other for PTPRC (HEX),with almost no yellow multiplexed drops that would be indicative of amultiplexed signal, as shown in FIG. 29, Panel B. This demonstrated thatcross-contamination during picoinjection is rare.

To measure the precise rate of cross-contamination, automated dropletdetection software was used to analyze thousands of drops, FIG. 30,Panel A, and the results were plotted as a percentage of the totalnumber of TaqMan® positive drops, FIG. 30, Panel B. A total of 5771TaqMan® positive control drops and 7329 TaqMan® positive picoinjecteddrops were analyzed from three independent experimental replicates. Forthe control drops, in which the Raji and PC3 RNA were combined, amultiplexing rate 44% (+/−9.26) was observed. By contrast, for thepicoinjected drops, only 0.31% (+/−0.14) multiplexed drops wereobserved, as shown in FIG. 30, Panel B. Hence, with picoinjection, therewas some multiplexing, although the rate was so low it cannot be ruledout as resulting from other sources of RNA transfer, such as merger ofdrops during thermocycling or transport of RNA between dropletinterfaces.

The dual population experiments in which the drops were picoinjectedimmediately after being formed allowed for the estimation of the preciseamount of cross-contamination, but in most actual implementations ofpicoinjection for biological assays, the drops will be formed on onedevice, removed offline for incubation or thermocycling, and thenreinjected into another device for picoinjection. To demonstrate thatpicoinjection is effective for digital RT-PCR reactions performed underthese conditions, and to estimate the rate of cross contamination, adual population of drops was again created, but this time the drops werepulled offline and stored in a 1 mL syringe before reinjecting andpicoinjecting them. Just as before, it was observed that nearly alldrops were pure green or red, indicating minimal cross contamination, asshown in FIG. 31. However, some drops with a multiplexed signal werealso observed, as shown by the rare yellow drops in the image. In thisexperiment, the multiplexing rate was 1%, higher than with the dropsthat were picoinjected immediately after formation. Whilecross-contamination at the picoinjector cannot be ruled out, it issuspected that the higher multiplexing rate was the result of merger ofdrops during offline storage and reinjection, during which the drops maybe subjected to dust, air, and shear forces that can increase thechances for merger. This is supported by the observation that duringreinjection of the emulsion there were occasional large merged drops,and also that the picoinjected emulsion was somewhat polydisperse, asshown in FIG. 31. Nevertheless, even under these rough conditions, thevast majority of drops displayed no multiplexing, indicating that theyretained their integrity as distinct reactors.

From these studies, it was demonstrated that picoinjection is compatiblewith droplet digital RT-PCR and affords single RNA molecule detectionrates equivalent to workflows not incorporating picoinjection. Thisshowed that picoinjection is compatible with reactions involving commonbiological components, like nucleic acids, enzymes, buffers, and dyes.It was also observed that there was negligible transfer of materialbetween drops during picoinjection. These results support picoinjectionas a powerful and robust technique for adding reagents to drops forultrahigh-throughput biological assays.

Example 6: Single Cell RT-PCR Microfluidic Device

FIG. 32 shows one embodiment of a single cell RT-PCR microfluidic deviceas provided herein. The cells of interested were first encapsulated indrops with lysis reagent including proteases and detergents andincubated offline. These drops were then introduced into this device andspaced by oil using an input microchannel and a flow focus drop makerfor introducing microdroplets (Panel A). In a pairing microchannel, thespaced drops were then paired with large drops containing a dilutionbuffer that were created by a dilution buffer drop maker in fluidiccommunication with the pairing microchannel (Panel B). The big and smalldrops were then merged in a merging microchannel with an electric field(Panel C), adding the contents of the small drop to the large drop. Themerged drops passed through mixing microchannels and then a smallportion was sampled from them by a drop sampler (Panel D). The smallportion was then passed by a picoinjection microchannel where the smallportion was then picoinjected with the RT-PCR reagent (Panel E). Thedrops were then thermocycled for the RT-PCR reaction.

This system facilitated single cell RT-PCR because it allowed for theperformance of the cell lysis and protein digestion in one step (notshown) and subsequent dilution of the lysate in the drop prior toaddition of the RT-PCR reagent. Without the dilution, the lysate couldhave inhibited the RT-PCR reaction.

The device worked robustly, at least in part, because the timing of eachmicrofluidic component was set by the periodicity of the large dropmaker making the dilution drops. Without this periodic drop formation,the device might operate less stably and potentially producepolydisperse drops.

Example 7: Testing of Microfluidic Droplet Forming Devices UtilizingChannels Including Ridges

T-junction drop makers with and without channel ridges positioneddownstream of the T-junction were tested to determine the effect ofincluding such ridges on droplet formation performance. The channelwidths were about 30 microns and the width of the ridge peaks were fromabout 5 to about 10 microns. See FIG. 33.

PDMS microfluidic devices were prepared generally as described hereinand plasma treated for 10 seconds. The flow rate ratio was monitored,wherein the sum (Q_(sum)) of individual flow rates (Q_(oil))+(Q_(aq))was approximately 1000 μl/hr, and the ratio (R)=Q_(aq)/Q_(sum), anddroplet formation was visualized.

As the flow rate ratio was increased for the device lacking ridges, thedrop maker stopped forming drops and instead formed a long jet. Withoutintending to be bound by any particular theory, it is believe that thiswas due to the jet wetting the channel walls and adhering, preventingthe formation of drops. See FIG. 33, left side. For the device whichincluded the ridges, the ridges successfully trapped oil near the walls,making it difficult for the aqueous phase to wet. This allowed thedevice to form drops at much higher flow rate ratios before iteventually wet at R=0.9. This demonstrated that the ridges allow thedrop maker to function over a much wider range than would be possiblewithout the ridges. The top and bottom sets of images in FIG. 33correspond to experiments performs with different devices. When theexperiment was performed with the first pair of devices, a 21-foldincrease in maximum Q_(aq)/Q_(oil) was achieved. When the sameexperiment was performed with a second set of devices, an 8-foldincrease in maximum Q_(aq)/Q_(oil) was achieved. This discrepancy may beattributed to experimental variability because the wetting propertiesthat lead to jetting are somewhat unpredictable, hysteretic, and proneto variability.

Example 8: Fabrication and Testing of Liquid Electrodes

Many microfluidic devices utilize metal electrodes to create electricfields when such fields are called for in a particular microfluidicdevice application. However, there may be disadvantages to using suchmetal electrodes including an increased number of fabrication steps andthe potential for failure of the electrodes.

Advantageously, the present disclosure describes the fabrication and useof liquid electrodes, which simplify the fabrication process and providesimilar and/or improved capabilities relative to metal electrodes.

FIG. 34 provides an overview of an exemplary liquid electrodefabrication method. Initially, an SU-8 photoresist master was fabricatedon an Si wafer (A). PDMS was then cast, degassed and cured (B). Inletports were punched in the PDMS, and the PDMS was bonded to a glass slide(C). Finally, the channel was filled with a NaCl solution. FIG. 35provides a sequence of three images taken at different times as anelectrode channel was being filled with salt water (time course proceedsfrom left to right). The salt water was introduced into the inlet of thechannel and pressurized, causing it to slowly fill the channel. The airthat was originally in the channel was pushed into the PDMS so that, bythe end, it was entirely filled with liquid.

Electric field lines for various liquid electrode configurations weresimulated as shown in FIG. 36. The simulations are of positive andground electrodes showing equipotential lines for three differentgeometries.

The liquid electrodes were capable of merging drops through applicationof an electric field as shown in FIG. 37, which provides two images of adroplet merger device that merges large drops with small drops utilizingliquid electrodes. To merge the drops, an electric field was appliedusing a salt-water electrode. When the field was off, no merger occurred(right) and when it was on, the drops merged (left).

Example 9: PCR Analysis and FACS Sorting of Azopira/E. coli Mixture

Two different species of microbes, Azospira and E. coli. Wereencapsulated in microdrops. In-droplet PCR was performed using TaqMan®and primers for Azospira and/or E. coli. FIG. 52 provides images showingdrops in which a TaqMan® PCR reaction was performed with encapsulatedAzospira. The upper images correspond to a reaction in which a 110 bpamplicon was produced, whereas the lower images to a 147 bp amplicon.FIG. 53 shows a picture of a gel testing 16S primers for Azospira and E.coli. The gel shows the bands corresponding to the amplicons of twoTaqMan® PCR reactions, one for a 464 bp amplicon and one for a 550 bpamplicon. FIG. 54 provides a picture of a gel validating that thein-droplet PCR reactions can be multiplexed by adding multiple primersets to a sample containing bacteria. FIG. 55 shows results for anexperiment where the TaqMan® reaction had primers and probes only forAzospira, so only the drops containing one of these microbes underwentamplification and became fluorescent, while the empty drops or the oneswith E. coli remained dim. The emulsion was then encapsulated intodouble emulsions using a microfluidic device and sorted on FACS. Theplots to the right in FIG. 55 show the FACS data. The upper plot showsthe scattering cross section plotted as a function of the dropfluorescence. Based on this, a population was gated out by drawingboundaries (shown above), and this population was sorted based on thedrop intensity. The gating allowed erroneous events due to small oildrops or dust to be discarded. When looking at only the doubleemulsions, the population had two distinct peaks which corresponded tothe fluorescent and non-fluorescent drops, shown in the lower histogram.An attempt to re-amplify the amplicons created during the in-dropletPCRs was unsuccessful, potentially due to their chemical structure sincethey may contain analogue bases or due to an inhibitory effect of thecarrier oil.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention being withoutlimitation to such specifically recited examples and conditions.Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

What is claimed is:
 1. A method of amplifying a target polynucleotide,the method comprising: (a) contacting on a microfluidic device abiological sample comprising a cell containing a target polynucleotidewith lysis reagents in a microdroplet, the lysis reagents comprising anenzyme having protease activity, wherein the microdroplet isencapsulated in an immiscible carrier fluid; (b) incubating themicrodroplet containing the sample and the lysis reagents comprising theenzyme having protease activity for a period of time sufficient to allowthe cell to lyse and form a cell lysate and the protease to digestinhibitory proteins; (c) incubating the microdroplet containing the celllysate and the enzyme having the protease activity at a temperaturesufficient to inactivate the enzyme having protease activity; (d) addingto the microdroplet containing the cell lysate, amplification reagentsto form a microdroplet containing the cell lysate and the amplificationreagents in the immiscible carrier fluid; and (e) amplifying the targetpolynucleotide within the microdroplet containing the cell lysate andthe amplification reagents; wherein the method does not comprise a stepof selectively removing reagents from the microdroplet containing thecell lysate and amplification reagents prior to step (e).
 2. The methodof claim 1, wherein the enzyme is a protease.
 3. The method of claim 1,wherein the enzyme is proteinase K.
 4. The method of claim 1, whereinthe microdroplet containing the cell lysate and the amplificationreagents has a volume of 0.001 to 1000 picoliters.
 5. The method ofclaim 1, wherein the microdroplet containing the cell lysate and theamplification reagents has a diameter of between 0.1 microns and 1000microns.
 6. The method of claim 1, wherein the microdroplet containingthe sample contains a single cell.
 7. The method of claim 1, wherein thestep (d) comprises (i) contacting the microdroplet containing the celllysate with a continuous stream of fluid comprising the amplificationreagents, and (ii) forming the microdroplet containing the cell lysateand the amplification reagents from a portion of the continuous streamof fluid and the microdroplet containing the cell lysate.
 8. The methodof claim 1, wherein the amplification comprises reverse transcription toproduce a reverse transcription product.
 9. The method of claim 1,wherein the amplification comprises polymerase chain reaction.
 10. Themethod of claim 1, further comprising detecting an amplification productof the target polynucleotide.
 11. The method of claim 10, whereindetecting the amplification product of the target polynucleotidecomprises determining a sequence of the amplification product.
 12. Themethod of claim 10, wherein detecting the amplification product of thetarget polynucleotide comprises forming a double-emulsion comprising themicrodroplet containing amplification product and sorting thedouble-emulsion based on at least one of droplet size and fluorescence.13. The method of claim 10, further comprising the step of sorting themicrodroplet based on results of detecting the amplification product ofthe target polynucleotide.
 14. The method of claim 10, furthercomprising determining a number of microdroplets containing the targetpolynucleotide based on results of detecting the amplification productof the target polynucleotide.
 15. The method of claim 10, furthercomprising determining percentage of microdroplets containing the targetpolynucleotide based on results of detecting the amplification productof the target polynucleotide.
 16. The method of claim 10, whereinamplifying the target polynucleotide comprises extension of a primercomprising a capture sequence and detecting the target polynucleotidecomprises detecting the capture sequence in the amplification product.17. The method of claim 16, wherein detecting presence of the capturesequence comprises introducing a fluorescent bead into the microdropletcontaining the amplification product, wherein the fluorescent beadcomprises a nucleotide sequence complementary to the capture sequence.18. The method of claim 1, wherein at least one of the steps (d) and (e)are performed under microfluidic control.
 19. The method of claim 1,wherein step (d) is performed under microfluidic control.
 20. The methodof claim 1, wherein the step (d) comprises droplet coalescence.
 21. Themethod of claim 1, wherein the step (d) comprises picoinjection.
 22. Themethod of claim 1, further comprising sorting prior to lysing the samplecontaining target polynucleotide.
 23. The method of claim 1, furthercomprising sorting between step (a) and step (d).